Composition of reverse transcriptases and mutants thereof

ABSTRACT

The invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity and to methods of producing, amplifying or sequencing nucleic acid molecules using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and use of such nucleic acid molecules to produce desired polypeptide. The invention also relates to methods for producing Avian Sarcoma-Leukosis Virus (ASLV) RT subunits, in particular, Avian Myeloblastosis Virus (AMV) RTs, to isolated nucleic acid molecules encoding ASLV RT subunits, and to ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant RT holoenzymes, particularly ASLV RTs, methods for producing these RTs and to RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, and ASLV RTs of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/064,057, filed Apr. 22, 1998, which claims the benefit of U.S.Provisional Application No. 60/044,589, filed Apr. 22, 1997, and60/049,874, filed Jun. 17, 1997, the disclosures of which are entirelyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention is in the fields of molecular and cellularbiology. The invention is generally related to reverse transcriptaseenzymes and methods for the reverse transcription of nucleic acidmolecules, especially messenger RNA molecules. Specifically, theinvention relates to compositions comprising mixtures of reversetranscriptase enzymes, and to methods of producing, amplifying orsequencing nucleic acid molecules (particularly cDNA molecules) usingthese reverse transcriptase enzymes or compositions. The invention alsorelates to nucleic acid molecules produced by these methods and to theuse of such nucleic acid molecules to produce desired polypeptides. Theinvention also concerns kits comprising such compositions.

BACKGROUND OF THE INVENTION

cDNA and cDNA Libraries

In examining the structure and physiology of an organism, tissue orcell, it is often desirable to determine its genetic content. Thegenetic framework of an organism is encoded in the double-strandedsequence of nucleotide bases in the deoxyribonucleic acid (DNA) which iscontained in the somatic and germ cells of the organism. The geneticcontent of a particular segment of DNA, or gene, is only manifested uponproduction of the protein which the gene encodes. In order to produce aprotein, a complementary copy of one strand of the DNA double helix (the“coding” strand) is produced by polymerase enzymes, resulting in aspecific sequence of ribonucleic acid (RNA). This particular type ofRNA, since it contains the genetic message from the DNA for productionof a protein, is called messenger RNA (mRNA).

Within a given cell, tissue or organism, there exist myriad mRNAspecies, each encoding a separate and specific protein. This factprovides a powerful tool to investigators interested in studying geneticexpression in a tissue or cell— mRNA molecules may be isolated andfurther manipulated by various molecular biological techniques, therebyallowing the elucidation of the full functional genetic content of acell, tissue or organism.

One common approach to the study of gene expression is the production ofcomplementary DNA (cDNA) clones. In this technique, the mRNA moleculesfrom an organism are isolated from an extract of the cells or tissues ofthe organism. This isolation often employs solid chromatographymatrices, such as cellulose or agarose, to which oligomers of thymidine(T) have been complexed. Since the 3′ termini on most eukaryotic mRNAmolecules contain a string of adenosine (A) bases, and since A binds toT, the mRNA molecules can be rapidly purified from other molecules andsubstances in the tissue or cell extract. From these purified mRNAmolecules, cDNA copies may be made using the enzyme reversetranscriptase (RT), which results in the production of single-strandedcDNA molecules. The single-stranded cDNAs may then be converted into acomplete double-stranded DNA copy (ie., a double-stranded cDNA) of theoriginal mRNA (and thus of the original double-stranded DNA sequence,encoding this mRNA, contained in the genome of the organism) by theaction of a DNA polymerase. The protein-specific double-stranded cDNAscan then be inserted into a plasmid or viral vector, which is thenintroduced into a host bacterial, yeast, animal or plant cell. The hostcells are then grown in culture media, resulting in a population of hostcells containing (or in many cases, expressing) the gene of interest.

This entire process, from isolation of mRNA to insertion of the cDNAinto a plasmid or vector to growth of host cell populations containingthe isolated gene, is termed “cDNA cloning.” If cDNAs are prepared froma number of different mRNAs, the resulting set of cDNAs is called a“cDNA library,” an appropriate term since the set of cDNAs represents a“population” of genes comprising the functional genetic informationpresent in the source cell, tissue or organism. Genotypic analysis ofthese cDNA libraries can yield much information on the structure andfunction of the organisms from which they were derived.

Retroviral Reverse Transcriptase Enzymes

Three prototypical forms of retroviral RT have been studied thoroughly.Moloney Murine Leukemia Virus (M-MLV) RT contains a single subunit of 78kDa with RNA-dependent DNA polymerase and RNase H activity. This enzymehas been cloned and expressed in a fully active form in E. coli(reviewed in Prasad, V. R., Reverse Transcriptase, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press, p. 135 (1993)). HumanImmunodeficiency Virus (HIV) RT is a heterodimer of p66 and p51 subunitsin which the smaller subunit is derived from the larger by proteolyticcleavage. The p66 subunit has both a RNA-dependent DNA polymerase and anRNase H domain, while the p51 subunit has only a DNA polymerase domain.Active HIV p66/p51 RT has been cloned and expressed successfully in anumber of expression hosts, including E. coli (reviewed in Le Grice, S.F. J., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory press, p. 163 (1993)). Within the HIV p66/p51heterodimer, the 51-kD subunit is catalytically inactive, and the 66-kDsubunit has both DNA polymerase and RNase H activity (Le Grice, S. F.J., et al., EMBO Journal 10:3905 (1991), Hostomsky, Z., et al., J.Virol. 66:3179 (1992)). Avian Sarcoma-Leukosis Virus (ASLV) RT, whichincludes but is not limited to Rous Sarcoma Virus (RSV) RT, AvianMyeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) HelperVirus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT,Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, AvianSarcoma Virus UR2 Helper Virus UR2AV RT, Avian Sarcoma Virus Y73 HelperVirus YAV RT, Rous Associated Virus (RAV) RT, and MyeloblastosisAssociated Virus (MAV) RT, is also a heterodimer of two subunits, α(approximately 62 kDa) and β (approximately 94 kDa), in which a isderived from β by proteolytic cleavage (reviewed in Prasad, V. R.,Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1993), p. 135). ASLV RT can exist in two additionalcatalytically active structural forms, ββ and α (Hizi, A. and Joklik, W.K., J. Biol. Chem. 252: 2281 (1977)). Sedimentation analysis suggestsαβ0 and ββ are dimers and that the α form exists in an equilibriumbetween monomeric and dimeric forms (Grandgenett, D. P., et al., Proc.Nat. Acad. Sci. USA 70: 230 (1973); Hizi, A. and Joklik, W. K., J. Biol.Chem. 252: 2281 (1977); and Soltis, D. A. and Skalka, A. M., Proc. Nat.Acad. Sci. USA 85: 3372 (1988)). The ASLV αβ and ββ RTs are the onlyknown examples of retroviral RT that include three different activitiesin the same protein complex: DNA polymerase, RNase H, and DNAendonuclease (integrase) activities (reviewed in Skalka, A. M., ReverseTranscriptase, Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress (1993), p. 193). The α form lacks the integrase domain andactivity.

Various forms of the individual subunits of ASLV RT have been cloned andexpressed. These include a 98-kDa precursor polypeptide that is normallyprocessed proteolytically to β and a 4-kDa polypeptide removed from theβ carboxy end (Alexander, F., et al., J. Virol. 61: 534 (1987) andAnderson, D. et al., Focus 17:53 (1995)), and the mature β subunit(Weis, J. H. and Salstrom, J. S., U.S. Pat. No. 4,663,290 (1987); andSoltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA 85:3372(1988)). Heterodimeric RSV αβ RT has also been purified from E. colicells expressing a cloned RSV β gene (Chernov, A. P., et al., BiomedSci. 2:49 (1991)). However, there have been no reports heretofore of thesimultaneous expression of cloned ASLV RT α and β genes resulting in theformation of heterodimeric αβ RT.

Reverse Transcription Efficiency

As noted above, the conversion of mRNA into cDNA by RT-mediated reversetranscription is an essential step in the study of proteins expressedfrom cloned genes. However, the use of unmodified RT to catalyze reversetranscription is inefficient for at least two reasons. First, RTsometimes destroys an RNA template before reverse transcription isinitiated, primarily due to the activity of intrinsic RNase H activitypresent in RT. Second, RT often fails to complete reverse transcriptionafter the process has been initiated (Berger, S. L., et al.,Biochemistry 22:2365-2372 (1983); Krug, M. S., and Berger, S. L., Meth.Enzymol. 152:316 (1987)). Removal of the RNase H activity of RT caneliminate the first problem and improve the efficiency of reversetranscription (Gerard, G. F., et al., FOCUS 11(4):60 (1989); Gerard, G.F., et al., FOCUS 14(3):91 (1992)). However RTs, including those formslacking RNase H activity (“RNase H⁻” forms), still tend to terminate DNAsynthesis prematurely at certain secondary structural (Gerard, G. F., etal., FOCUS 11(4):60 (1989); Myers, T. W., and Gelfand, D. H.,Biochemistry 30:7661 (1991)) and sequence (Messer, L. I., et al., Virol146:146 (1985)); Abbotts, J., et al., J. Biol. Chem. 268:10312-10323(1993)) barriers in nucleic acid templates.

Even in the most efficient reverse transcription systems availabletoday, which use RNase H⁻M-MLV RT, yields of total cDNA productgenerally do not exceed 50% of input mRNA and the fraction of theproduct that is full-length does not exceed 50%. The secondarystructural and sequence barriers in the mRNA template, which asdescribed above can give rise to these limitations, occur frequently athomopolymer stretches (Messer, L. I., et al., Virol. 146:146 (1985);Huber, H. E., et al., J. Biol. Chem. 264:4669-4678 (1989); Myers, T. W.,and Gelfand, D. H., Biochemistry 30:7661 (1991)), are more oftensequence rather than secondary structural barriers (Abbotts, J., et al.,J. Biol. Chem. 268:10312-10323 (1993)), and are often distinct fordifferent RTs (Abbotts, J., et al., J. Biol. Chem. 268:10312-10323(1993)). If these barriers could be overcome, yield of total andfull-length cDNA product in reverse transcription reactions could beincreased.

SUMMARY OF THE INVENTION

The present invention provides reverse transcriptase enzymes,compositions comprising such enzymes and methods useful in overcomingthe above-described cDNA length limitations. In general, the inventionprovides compositions for use in reverse transcription of a nucleic acidmolecule comprising two or more different polypeptides having reversetranscriptase activity. Such compositions may further comprise one ormore nucleotides, a suitable buffer, and/or one or more DNA polymerases.The compositions of the invention may also comprise one or moreoligonucleotide primers. Each reverse transcriptase used in thecompositions of the invention may have a different transcription pausesite on a given mRNA molecule. The reverse transcriptases in thesecompositions preferably are reduced or substantially reduced in RNase Hactivity, and most preferably are enzymes selected from the groupconsisting of Moloney Murine Leukemia Virus (M-MLV) H⁻ reversetranscriptase, Rous Sarcoma Virus (RSV) H⁻ reverse transcriptase, AvianMyeloblastosis Virus (AMV) H⁻ reverse transcriptase, Rous AssociatedVirus (RAV) H⁻ reverse transcriptase, Myeloblastosis Associated Virus(MAV) H⁻ reverse transcriptase and Human Immunodeficiency Virus (HIV) H⁻reverse transcriptase or other ASLV H⁻ reverse transcriptases. Inpreferred compositions, the reverse transcriptases are present atworking concentrations.

The invention is also directed to methods for making one or more nucleicacid molecules, comprising mixing one or more nucleic acid templates(preferably one or more RNA templates and most preferably one or moremessenger RNA templates) with two or more polypeptides having reversetranscriptase activity and incubating the mixture under conditionssufficient to make a first nucleic acid molecule or moleculescomplementary to all or a portion of the one or more nucleic acidtemplates. In a preferred embodiment, the first nucleic acid molecule isa single-stranded cDNA. Nucleic acid templates suitable for reversetranscription according to this aspect of the invention include anynucleic acid molecule or population of nucleic acid molecules(preferably RNA and most preferably mRNA), particularly those derivedfrom a cell or tissue. In a preferred aspect, a population of mRNAmolecules (a number of different mRNA molecules, typically obtained fromcells or tissue) are used to make a cDNA library, in accordance with theinvention. Preferred cellular sources of nucleic acid templates includebacterial cells, fungal cells, plant cells and animal cells.

The invention also concerns methods for making one or moredouble-stranded nucleic acid molecules. Such methods comprise (a) mixingone or more nucleic acid templates (preferably RNA or mRNA, and morepreferably a population of mRNA templates) with two or more polypeptideshaving reverse transcriptase activity; (b) incubating the mixture underconditions sufficient to make a first nucleic acid molecule or moleculescomplementary to all or a portion of the one or more templates; and (c)incubating the first nucleic acid molecule under conditions sufficientto make a second nucleic acid molecule or molecules complementary to allor a portion of the first nucleic acid molecule or molecules, therebyforming one or more double-stranded nucleic acid molecules comprisingthe first and second nucleic acid molecules. Such methods may includethe use of one or more DNA polymerases as part of the process of makingthe one or more double-stranded nucleic acid molecules. The inventionalso concerns compositions useful for making such double-strandednucleic acid molecules. Such compositions comprise two or more reversetranscriptases and optionally one or more DNA polymerases, a suitablebuffer and one or more nucleotides.

The invention also relates to methods for amplifying a nucleic acidmolecule. Such amplification methods comprise mixing the double-strandednucleic acid molecule or molecules produced as described above with oneor more DNA polymerases and incubating the mixture under conditionssufficient to amplify the double-stranded nucleic acid molecule. In afirst preferred embodiment, the invention concerns a method foramplifying a nucleic acid molecule, the method comprising (a) mixing oneor more nucleic acid templates (preferably one or more RNA or mRNAtemplates and more preferably a population of mRNA templates) with twoor more different polypeptides having reverse transcriptase activity andwith one or more DNA polymerases and (b) incubating the mixture underconditions sufficient to amplify nucleic acid molecules complementary toall or a portion of the one or more templates. Preferably, the reversetranscriptases are reduced or substantially reduced in RNase H activityand the DNA polymerases comprise a first DNA polymerase having 3′exonuclease activity and a second DNA polymerase having substantiallyreduced 3′ exonuclease activity. The invention also concernscompositions comprising two or more reverse transcriptases and one ormore DNA polymerases for use in amplification reactions. Suchcompositions may further comprise one or more nucleotides and a buffersuitable for amplification. The compositions of the invention may alsocomprise one or more oligonucleotide primers.

In accordance with the invention, at least two, at least three, at leastfour, at least five, at least six, or more, reverse transcriptases maybe used. Preferably, two to six, two to five, two to four, two to three,and most preferably two, reverse transcriptases are used in thecompositions and methods of the invention. Such multiple reversetranscriptases may be added simultaneously or sequentially in any orderto the compositions or in the methods of the invention. Alternatively,multiple different reactions with different enzymes may be performedseparately and the reaction products may be mixed. Thus, the inventionrelates to the synthesis of the nucleic acid molecules by the methods ofthe invention in which multiple reverse transcriptases are usedsimultaneously or sequentially or separately. In particular, theinvention relates to a method of making one or more nucleic acidmolecules comprising incubating one or more nucleic acid templates(preferably one or more RNA templates or mRNA templates, and morepreferably a population of mRNA templates) with a first reversetranscriptase under conditions sufficient to make one or more nucleicacid molecules complementary to all or a portion of the one or moretemplates. In accordance with the invention, the one or more nucleicacid molecules (including mRNA templates and/or synthesized nucleic acidmolecules) may be incubated with a second reverse transcriptase underconditions sufficient to make additional nucleic acid moleculescomplementary to all or a portion of the templates or to increase thelength of the previously made nucleic acid molecules. In accordance withthe invention, this procedure may be repeated any number of times withthe same or different reverse transcriptases of the invention. Forexample, the first and second reverse transcriptases may be the same ordifferent. Furthermore, the first and third reverse transcriptases (inaspects of the invention where the procedure is repeated three timesusing a first, second, and third reverse transcriptase) may be the samewhile the second reverse transcriptase may be different from the firstand the third reverse transcriptase. Thus, any combination of the sameand/or different reverse transcriptases may be used in this aspect ofthe invention. Preferably, when multiple reverse transcriptases areused, at least two reverse transcriptases are different.

In a related aspect of the invention, the reverse transcriptase used inthe reaction may retain all or a portion of its activity duringsubsequent reaction steps. Alternatively, the reverse transcriptase usedin the reaction may be inactivated by any method prior to incubationwith additional reverse transcriptases. Such an inactivation may includebut is not limited to heat inactivation, organic extraction (e.g., withphenol and/or chloroform), ethanol precipitation and the like.

The synthesized nucleic acid molecules made by simultaneous orsequential or separate addition of reverse transcriptases may then beused to make double stranded nucleic acid molecules. Such synthesizednucleic acid molecules serve as a template which when incubated underappropriate conditions (e.g., preferably in the presence of one or moreDNA polymerases) make nucleic acid molecules complementary to all or aportion of the synthesized nucleic acid molecules, thereby forming anumber of double stranded nucleic acid molecules. The double strandedmolecules may then be amplified in accordance with the invention.

The invention is also directed to nucleic acid molecules (particularlysingle- or double-stranded cDNA molecules) or amplified nucleic acidmolecules produced according to the above-described methods and tovectors (particularly expression vectors) comprising these nucleic acidmolecules or amplified nucleic acid molecules.

The invention is also directed to recombinant host cells comprising theabove-described nucleic acid molecules, amplified nucleic acid moleculesor vectors. Preferred such host cells include bacterial cells, yeastcells, plant cells and animal cells (including insect cells andmammalian cells).

The invention is further directed to methods of producing a polypeptidecomprising culturing the above-described recombinant host cells andisolating the polypeptide, and to a polypeptide produced by suchmethods.

The invention also concerns methods for sequencing one or more nucleicacid molecules using the compositions or enzymes of the invention. Suchmethods comprise (a) mixing one or more nucleic acid molecules (e.g.,one or more RNA or DNA molecules) to be sequenced with one or moreprimers, one or more polypeptides having reverse transcriptase activity,one or more nucleotides and one or more terminating agents, such as oneor more dideoxynucleoside triphosphates; (b) incubating the mixtureunder conditions sufficient to synthesize a population of nucleic acidmolecules complementary to all or a portion of the one or more nucleicacid molecules to be sequenced; and (c) separating the population ofnucleic acid molecules to determine the nucleotide sequence of all or aportion of the one or more nucleic acid molecules to be sequenced. Inthese sequencing methods of the invention, the one or more polypeptideshaving reverse transcriptase activity may be added simultaneously,sequentially, or separately to the reaction mixtures as described above.

The invention is also directed to kits for use in the methods of theinvention. Such kits can be used for making, sequencing or amplifyingnucleic acid molecules (single- or double-stranded). The kits of theinvention comprise a carrier, such as a box or carton, having in closeconfinement therein one or more containers, such as vials, tubes,bottles and the like. In the kits of the invention, a first containercontains one or more of the reverse transcriptase enzymes (preferablyone or more such enzymes that are reduced or substantially reduced inRNase H activity) or one or more of the compositions of the invention.In another aspect, the kit may contain one or more containers comprisingtwo or more, three or more, four or more, five or more, six or more, andthe like, reverse transcriptases, preferably one or more containerscomprising two to six, two to five, two to four, two to three, or morepreferably two, reverse transcriptases. The kits of the invention mayalso comprise, in the same or different containers, one or more DNApolymerase (preferably thermostable DNA polymerases), a suitable bufferfor nucleic acid synthesis and one or more nucleotides. Alternatively,the components of the composition may be divided into separatecontainers (e.g., one container for each enzyme). In preferred kits ofthe invention, the reverse transcriptases are reduced or substantiallyreduced in RNase H activity, and are most preferably selected from thegroup consisting of M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV H⁻ reversetranscriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase. In additional preferred kits of the invention, theenzymes (reverse transcriptases and/or DNA polymerases) in thecontainers are present at working concentrations.

The invention also relates to methods of producing RSV reversetranscriptase (and/or subunits thereof). In particular, the inventionrelates to methods for producing RSV reverse transcriptase (and/orsubunits thereof) containing RNase H activity, to methods for producingRSV reverse transcriptase (and/or subunits thereof) that is reduced orsubstantially reduced in RNase H activity, and to RSV reversetranscriptases (and/or subunits thereof) produced by such methods.

The invention further relates to methods for using such reversetranscriptases and to kits comprising such reverse transcriptases. Inparticular, the RSV reverse transcriptases (and/or subunits thereof) ofthe invention may be used in methods of sequencing, amplification andproduction (via, e.g., reverse transcription) of nucleic acid molecules.

The invention also relates to methods of producing AMV reversetranscriptase (and/or subunits thereof). In particular, the inventionrelates to methods for producing AMV reverse transcriptase (and/orsubunits thereof) containing RNase H activity, to methods for producingAMV reverse transcriptase (and/or subunits thereof) that is reduced orsubstantially reduced in RNase H activity, and to AMV reversetranscriptases (and/or subunits thereof) produced by such methods.

The invention further relates to methods for using such reversetranscriptases and to kits comprising such reverse transcriptases. Inparticular, the AMV reverse transcriptases (and/or subunits thereof ofthe invention may be used in methods of sequencing, amplification andproduction (via, e.g., reverse transcription) of nucleic acid molecules.

The invention also generally relates to methods of producing ASLVreverse transcriptases (and/or subunits thereof). In particular, theinvention relates to methods for producing ASLV reverse transcriptases(and/or subunits thereof) containing RNase H activity, to methods forproducing such ASLV reverse transcriptases that are reduced orsubstantially reduced in RNase H activity, and to ASLV reversetranscriptases produced by such methods.

The invention further relates to methods for using such reversetranscriptases and to kits comprising such reverse transcriptases. Inparticular, the ASLV reverse transcriptases (and/or subunits thereof) ofthe invention may be used in methods of sequencing, amplification andproduction (e.g., via reverse transcription) of nucleic acid molecules.

The invention further relates to methods for elevated- orhigh-temperature reverse transcription of a nucleic acid moleculecomprising (a) mixing one or more nucleic acid templates (preferably oneor more RNA molecules (e.g., one or more mRNA molecules or polyA+RNAmolecules, and more preferably a population of mRNA molecules) or one ormore DNA molecules) with one or more polypeptides having reversetranscriptase activity; and (b) incubating the mixture at a temperatureof 50° C. or greater and under conditions sufficient to make a firstnucleic acid molecule or molecules (such as a full length cDNA molecule)complementary to all or a portion of the one or more nucleic acidtemplates. In a preferred aspect, a population of mRNA molecules is usedto make a cDNA library at elevated or high temperatures. In anotheraspect, elevated- or high-temperature nucleic acid synthesis isconducted with multiple reverse transcriptases (i.e., two or more, threeor more, four or more, five or more, six or more, and the like, morepreferably two to six, two to five, two to four, two to three, and stillmore preferably two, reverse transcriptases), which may be added to thereaction mixture simultaneously or sequentially or separately asdescribed above. In preferred such methods, the mixture is incubated ata temperature of about 51° C. or greater, about 52° C. or greater, about53° C. or greater, about 54° C. or greater, about 55° C. or greater,about 56° C. or greater, about 57° C. or greater, about 58° C. orgreater, about 59° C. or greater, about 60° C. or greater, about 61° C.or greater, about 62° C. or greater, about 63° C. or greater, about 64°C. or greater, about 65° C. or greater, about 66° C. or greater, about67° C. or greater, about 68° C. or greater, about 69° C. or greater,about 70° C. or greater, about 71° C. or greater, about 72° C. orgreater, about 73° C. or greater, about 74° C. or greater, about 75° C.or greater, about 76° C. or greater, about 77° C. or greater, or about78° C. or greater; or at a temperature range of from about 50° C. toabout 75° C., about 51° C. to about 75° C., about 52° C. to about 75°C., about 53° C. to about 75° C., about 54° C. to about 75° C., about55° C. to about 75° C., about 50° C. to about 70° C., about 51° C. toabout 70° C., about 52° C. to about 70° C., about 53° C. to about 70°C., about 54° C. to about 70° C., about 55° C. to about 70° C., about55° C. to about 65° C., about 56° C. to about 65° C., about 56° C. toabout 64° C. or about 56° C. to about 62° C. The invention is alsodirected to such methods which further comprise incubating the firstnucleic acid molecule or molecules under conditions sufficient to make asecond nucleic acid molecule or molecules complementary to all orportion of the first nucleic acid molecule or molecules. According tothe invention, the first and second nucleic acid molecules produced bythese methods may be DNA molecules, and may form a double stranded DNAmolecule or molecules which may be a full length cDNA molecule ormolecules, such as a cDNA library. The one or more polypeptides havingreverse transcriptase activity that are used in these methods preferablyare reduced or substantially reduced in RNase H activity, and arepreferably selected from ASLV reverse transcriptases (and/or subunitsthereof) such as one or more subunits of AMV reverse transcriptaseand/or one or more subunits of RSV reverse transcriptase and/or one ormore subunits of MAV reverse transcriptase, and/or one or more subunitsof RAV reverse transcriptase, particularly wherein the subunits arereduced or substantially reduced in RNase H activity.

The invention also relates to kits for elevated- or high-temperaturenucleic acid synthesis, which may comprise one or more componentsselected from the group consisting of one or more reverse transcriptases(preferably one or more ASLV reverse transcriptases such as AMV or RSVreverse transcriptases (or one or more subunits thereof), and morepreferably one or more AMV or RSV reverse transcriptases (or one or moresubunits thereof) which are reduced or substantially reduced in RNase Hactivity), one or more nucleotides, one or more primers and one or moresuitable buffers.

The invention is also directed to nucleic acid molecules produced by theabove-described methods which may be full-length cDNA molecules, tovectors (particularly expression vectors) comprising these nucleic acidmolecules and to host cells comprising these vectors and nucleic acidmolecules.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following drawings and descriptionof the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-6 describe, in schematic form (with details omitted forclarity), the construction of the expression vector (pDABH⁻His) whichplaces the RSV RT α and β genes under control of insect viral promoters:

FIG. 1: pJD100, pAMP18, pAMP18N, pAMP1, pAMP1C., pAMP18NM and pAMP18B.

FIG. 2: M13, M13RT, pAMP18BH− and M13RTH−.

FIG. 3: pAMP1A.

FIG. 4: pDBH-Kpn, pDABH−, pDBH-KpnHis.

FIG. 5: pFastBac DUAL, pFastBac DUAL Nde, pDBH− and pDA.

FIG. 6: pDABH-His.

FIGS. 7-21 are more detailed maps of the plasmids described in FIGS.1-6:

FIG. 7: pJD100.

FIG. 8: pAMP18N.

FIG. 9: pAMP1C.

FIG. 10: pAMP18NM.

FIG. 11: pAMP18B.

FIG. 12: M13RT.

FIG. 13: M13RTH−.

FIG. 14: pAMP18BH−.

FIG. 15: pDBH−.

FIG. 16: pAMP1A.

FIG. 17: pDBH-Kpn.

FIG. 18: pDBH-KpnHis.

FIG. 19: pDA.

FIG. 20: pDABH−.

FIG. 21: pDABH-His.

FIGS. 22-25 describe, in schematic form (with details omitted forclarity), the construction of the expression vector (pDAMVAH−BH−)whichplaces the AMV RT α and β genes under control of insect viral promoters:

FIG. 22: Cloning of AMV RT gene from RNA; pSPORT8.

FIG. 23: Construction of a His-tagged AMV RT β gene; pAMVN, pAMVNM,pAMVNMH−, pAMVC and pAMVBH−.

FIG. 24: Construction of clones for the AMV RT α subunit by PCR; pAMVAand pAMVAH−.

FIG. 25: Construction of vectors comprising the AMV RT α and β genes;pD, pDAMVAH−, pDAMVA, pAMVH−BH−, pDAMVABH− and pJAMVBH−.

FIGS. 26-38 are more detailed maps of the plasmids described in FIGS.22-25:

FIG. 26: pAMVN.

FIG. 27: pAMVC.

FIG. 28: pAMVNM.

FIG. 29: pAMVNMH−.

FIG. 30: pAMVBH−.

FIG. 31: pAMVA.

FIG. 32: pAMVAH−.

FIG. 33: pFastBacDual (pD).

FIG. 34: pDAMVA.

FIG. 35: pDAMVAH−.

FIG. 36: pDAMVABH−.

FIG. 37: pJAMVBH−.

FIG. 38: pDAMVAH−BH−.

FIG. 39 is a semi-logarithmic graph demonstrating RT activities ofvarious RTs incubated for the times and at the temperatures indicated.

FIG. 40 is an autoradiograph of cDNA products synthesized by various RTsat the temperatures (° C.) indicated from 1.4-, 2.4-, 4.4- and 7.5-KbmRNAs over 50-minute reactions. M: ³²P-labeled 1 Kb DNA ladder.

FIG. 41 is an autoradiograph of cDNA products synthesized by RSV H⁻ RTand SS II RT at the temperatures (° C.) indicated from 1.4-, 2.4-, 4.4-,and 7.5-Kb mRNAs over 30-minute reactions. M: ³P-labeled 1 Kb DNAladder.

FIG. 42 is a graph of the amounts of full length cDNA synthesized bySSII and RSV H⁻ RT in FIG. 41 as a function of incubation temperature.

FIG. 43 is a restriction map of plasmid pBP-RT(PCR).

FIG. 44 is a restriction map of plasmid pBP-RT(ATG).

FIG. 45 is a restriction map of plasmid pBK-RT15(ATG).

FIG. 46 is a restriction map of plasmid pFBBH-His.

FIG. 47 is a restriction map of plasmid pJB-His.

FIG. 48 is a restriction map of plasmid pJBD110E-His.

FIG. 49 is a restriction map of plasmid pDAD110E.

FIG. 50 is a restriction map of plasmid pFBBD110E-His.

FIG. 51 is a restriction map of plasmid pDABHis.

FIG. 52 is a restriction map of plasmid pDABD110EHis.

FIG. 53 is a restriction map of plasmid pDAD110EBHis.

FIG. 54 is a restriction map of plasmid pDAD110EB110E.

DETAILED DESCRIPTION OF THE INVENTION

Overview

The present invention provides compositions and methods useful inovercoming the length limitations often observed during reversetranscription of nucleic acid molecules. Thus, the invention facilitatesthe production of full-length cDNA molecules not heretofore possible.

In general, the invention provides compositions for use in reversetranscription of a nucleic acid molecule comprising two or more, threeor more, four or more, five or more, six or more, and the like,different polypeptides having reverse transcriptase activity. Thecompositions of the invention preferably comprise two to six, two tofive, two to four, two to three, and more preferably comprise two,polypeptides having reverse transcriptase activity. The enzymes in thesecompositions are preferably present in working concentrations and arereduced or substantially reduced in RNase H activity, although mixturesof enzymes, some having RNase H activity and some reduced orsubstantially reduced in RNase H activity, may be used in thecompositions of the invention. Alternatively, the reverse transcriptasesused in the compositions of the invention may have RNase H activity.Preferred reverse transcriptases include M-MLV H⁻ reverse transcriptase,RSV H⁻ reverse transcriptase, AMV H⁻ reverse transcriptase, RAV H⁻reverse transcriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase or other ASLV H⁻ reverse transcriptases.

The invention is also directed to methods for reverse transcription ofone or more nucleic acid molecules comprising mixing one or more nucleicacid templates, which is preferably RNA or messenger RNA (mRNA) and morepreferably a population of rnRNA molecules, with two or morepolypeptides having reverse transcriptase activity (or with thecompositions of the invention) and incubating the mixture underconditions sufficient to make a nucleic acid molecule or moleculescomplementary to all or a portion of the one or more templates. Suchnucleic acid synthesis may be accomplished by sequential or simultaneousor separate addition of multiple reverse transcriptases. Preferably, twoor more, three or more, four or more, five or more, six or more, and thelike, reverse transcriptases are used, or a range of two to six, two tofive, two to four, two to three and more preferably two, reversetranscriptases are used. To make the nucleic acid molecule or moleculescomplementary to the one or more templates, a primer (e.g., an oligo(dT)primer) and one or more nucleotides are used for nucleic acid synthesisin the 3′ to 5′ direction. Nucleic acid molecules suitable for reversetranscription according to this aspect of the invention include anynucleic acid molecule, particularly those derived from a prokaryotic oreukaryotic cell. Such cells may include normal cells, diseased cells,transformed cells, established cells, progenitor cells, precursor cells,fetal cells, embryonic cells, bacterial cells, yeast cells, animal cells(including human cells), avian cells, plant cells and the like, ortissue isolated from a plant or an animal (e.g., human, cow, pig, mouse,sheep, horse, monkey, canine, feline, rat, rabbit, bird, fish, insect,etc.). Such nucleic acid molecules may also be isolated from viruses.

The invention further provides methods for amplifying or sequencing anucleic acid molecule comprising contacting the nucleic acid moleculewith two or more polypeptides having reverse transcriptase activity (orwith the compositions of the invention). Such reactions maybeaccomplished by sequential or simultaneous or separate addition of thetwo or more polypeptides having reverse transcriptase activity to thereaction mixtures. Preferred such methods comprise one or morepolymerase chain reactions (PCRs).

The invention also provides cDNA molecules or amplified nucleic acidmolecules produced according to the above-described methods, vectorsparticularly expression vectors) comprising these cDNA molecules oramplified nucleic acid molecules, and recombinant host cells comprisingsuch cDNA molecules, amplified nucleic acid molecules or vectors. Theinvention also provides methods of producing a polypeptide comprisingculturing these recombinant host cells and isolating the polypeptide,and provides a polypeptide produced by such methods.

The invention also provides kits for use in accordance with theinvention. Such kits comprise a carrier means, such as a box or carton,having in close confinement therein one or more container means, such asvials, tubes, bottles and the like, wherein the kit comprises, in thesame or different containers, two or more polypeptides having reversetranscriptase activity. The kits of the invention may also comprise, inthe same or different containers, one or more DNA polymerases, asuitable buffer and/or one or more nucleotides (such as deoxynucleosidetriphosphates (dNTPs)).

The invention also concerns a substantially pure RSV reversetranscriptase (RSV RT), which may or may not be reduced or substantiallyreduced in RNase H activity. Such RSV RTs may comprise one or moresubunits (or derivatives, variants, fragments or mutants thereof)selected from one or more α subunits, one or more β subunits, and one ormore βp4 subunits, any or all of which may or may not be reduced orsubstantially reduced in RNase H activity. In one preferred aspect ofthe invention, the RSV RT may comprise an a subunit reduced orsubstantially reduced in RNase H activity and a β subunit having RNase Hactivity (i.e., RSV αH⁻βH⁺ RT). In a preferred aspect of thisembodiment, the gene encoding the a subunit has been modified or mutatedto reduce RNase H activity while the gene encoding the β subunit has notbeen mutated or modified in this manner. Such mutations or modificationsare preferably made within the RNase H domain of the α subunit.Unexpectedly, the phenotype of this construct showed substantiallyreduced (i.e., approximately 5% of wildtype) RNase H activity. Inanother preferred aspect, the RSV RT may comprise an a subunit reducedor substantially reduced in RNase H activity and a β subunit alsoreduced or substantially reduced in RNase H activity (i.e., RSV αH⁻/βH⁻RT). In another preferred aspect, the RSV RT may comprise two βsubunits, either or both of which may or may not be reduced orsubstantially reduced in RNAse H activity (i.e., RSV βH⁻/βH⁻ RT; RSVβH⁻/βH⁺ RT; or RSV βH⁺/βH⁺ RT). In another preferred aspect, the RSV RTmay comprise a single a subunit, which may or may not be reduced orsubstantially reduced in RNAse H activity (i.e., RSV αH⁻ RT or RSV αH⁺RT). In another preferred aspect, the RSV RT may comprise one or moreβp4 subunits, any or all of which may or may not be reduced orsubstantially reduced in RNAse H activity (e.g., RSV βp4H⁻/βp4H⁻ RT; RSVβp4H⁻/βp4H⁺ RT; RSV βp4H⁺/βp4H⁺ RT; RSV αH⁻/βp4H⁺ RT; RSV αH⁺/βp4H⁺ RT;RSV αH⁻/βp4H⁻ RT; RSV αH⁺/βp4H⁻ RT; RSV βH⁻/βp4H⁺ RT; RSV βH⁻/βp4H⁻ RT;RSV βH⁺/βp4H⁻ RT; RSV βH⁺/βp4H⁺ RT; etc.). As will be recognized,derivatives, variants, fragments or mutants of any or all of the abovesubunits may also be used in accordance with the invention.

In a related aspect of the invention, where the RSV RTs comprise two ormore subunits (or derivatives, variants, fragments or mutants thereof)and preferably comprise two subunits (e.g., a dimer), at least one butpreferably not all of these subunits may be modified or mutated toreduce, substantially reduce or eliminate the polymerase activity of atleast one subunit (e.g., pol−). In a preferred aspect, for an RSV RTwhich comprises an α and β subunit, the β subunit has been modified ormutated (preferably by recombinant techniques) to reduce, substantiallyreduce or eliminate the polymerase activity while the polymeraseactivity of the a subunit has not been mutated or modified in thismanner. Preferably, the a subunit of such RSV RT has also been modifiedor mutated to reduce or substantially reduce RNase H activity while theβ subunit has not been mutated or modified in this manner. Such aconstruct may be designated αH−/βH+pol−. Any number of combinations ofsubunits can be prepared in which mutations or modifications are made inone subunit of a two subunit enzyme, and these constructs may becombined with other modifications or mutations, such as those whichreduce or substantially reduce RNase H activity. Illustrated examplesinclude but are not limited to RSV αH−/βH−pol−; RSV αH+/βH+pol−; RSVαH−pol−/βH+; RSV αH−pol−/βH−; RSV αH+pol−/βH+; RSV βH−/βH−pol−; RSVβH−/βH+pol−; RSV βH+/βH+pol−; RSV βp4H−/βp4H−pol−; RSV βp4H−/βp4H+pol−;RSV βp4H+/βp4H+pol−; RSV αH−/βp4H−pol−; RSV αH+/βp4H+pol−; RSVαH+/βp4H−pol−; RSV βH−/βp4H+pol−; RSV βH−/βp4H+pol−; RSV βH+/βp4H−pol−;etc. In a preferred aspect, the polymerase domain of the subunit ismodified or mutated (one or more point mutations, deletion mutationsand/or insertion mutations) by recombinant techniques. In anotheraspect, the nucleotide binding site of the polymerase domain is modifiedor mutated. In a preferred aspect, one or more acidic amino acids withinthe nucleotide binding site are substituted with different amino acids.Particularly preferred amino acids within the nucleotide binding sitefor mutation or modification include, but are not limited to, Asp¹⁰⁷,Leu¹⁰⁹, Lys¹⁰⁹, and Asp¹¹⁰, or the corresponding amino acid sequence.

The invention also relates to methods of producing the RSV RTs of theinvention, which methods comprise obtaining a host cell comprising anucleic acid sequence encoding one or more α subunits (or derivatives,variants, fragments or mutants thereof) and/or a nucleic acid sequenceencoding one or more β subunits (or derivatives, variants, fragments ormutants thereof) and/or a nucleic acid sequence encoding one or more βp4subunits (or derivatives, variants, fragments or mutants thereof, andculturing the host cell under conditions sufficient to produce the RSVRTs of the invention. The nucleic acid sequences encoding such αsubunit(s) and/or such β subunit(s) and/or such βp4 subunit(s) may becontained in the same vector or in different vectors. In accordance withthe invention, such α subunit(s) and/or β subunit(s) and/or βp4subunit(s) may be produced separately and mixed before or afterisolation of each subunit to form the RSV RTs of the invention.Alternatively, such α and/or β subunits and/or βp4 subunits may beexpressed simultaneously (i.e., co-expressed) in the same host cell,thereby producing an RSV RT comprising an α and a β subunit, an αsubunit alone, a β subunit alone, a βp4 subunit alone, a β subunit and aβp4 subunit, two β subunits, or two βp4 subunits, or derivatives,variants, fragments or mutants thereof. In a preferred aspect, the αsubunit (or derivatives, variants, fragments or mutants thereof) issimultaneously expressed in a host cell with the β subunit (orderivatives, variants, fragments or mutants thereof). In a relatedaspect of the invention, the β or βp4 subunits (or derivatives,fragments or mutants thereof) may be expressed in a host or host cellwhich accomplishes in vivo processing of some or all of such β or βp4subunits to form the corresponding α subunit. The presence of both the βand the α subunits allows in vivo formation of an RSV RT which comprisesan α and a β subunit. Such in vivo processing is preferably accomplishedby expressing the β and/or βp4 subunits (or derivatives, variants,fragments or mutants thereof) in a host cell, which may be prokaryoticor eukaryotic, having appropriate processing enzymes or proteins whichcleave such β or βp4 subunits to form a corresponding α subunit. Suchprocessing enzymes or proteins may be introduced and expressed in thehost system by recombinant means or may exist naturally in the hostsystem. Preferred hosts for in vivo processing include eukaryotic cellsor organelles such as yeast, fungi, plants, animals, insects, fish, andthe like. Recombinant systems (vectors, expression vectors, promoters,etc.) which allow cloning of the β or βp4 subunits (or derivatives,fragments or mutants thereof) for in vivo processing are well known toone of ordinary skill in the art. As noted above, any or all of the αand/or β and/or βp4 subunits of the RSV RT (or derivatives, variants,fragments or mutants thereof) produced by these recombinant techniquesmay be reduced or substantially reduced in RNase H activity.

These RSV RTs or subunits thereof may then be isolated from the hostcell, and may be substantially purified by any method of proteinpurification that will be familiar to those of ordinary skill in the art(e.g., chromatography, electrophoresis, dialysis, high-saltprecipitation, or combinations thereof). The invention also relates tokits comprising one or more of the RSV RTs of the invention.

The invention also concerns a substantially pure Avian MyeloblastosisVirus reverse transcriptase (AMV RT), which may or may not be reduced orsubstantially reduced in RNase H activity. Such AMV RTs may comprise oneor more subunits selected from one or more α subunits, one or more βsubunits, and one or more βp4 subunits (or derivatives, variants,fragments or mutants thereof), any or all of which may or may not bereduced or substantially reduced in RNase H activity. In one preferredaspect of the invention, the AMV RT may comprise an α subunit reduced orsubstantially reduced in RNase H activity and a β subunit having RNase Hactivity (i.e., AMV αH⁻ βH⁺ RT). In a particularly preferred aspect ofthis embodiment, the gene encoding the α subunit has been modified ormutated to reduce RNase activity (preferably within the RNase H domain)while the gene encoding the β subunit has not been modified or mutatedto affect RNase H activity. Unexpectedly, this construct demonstrates aphenotype in which the RNase H activity of the AMV RT comprising the αsubunit and the β subunit is substantially reduced in RNase H activity(i.e., approximately 5% of wildtype). In another preferred aspect, theAMV RT may comprise an α subunit reduced or substantially reduced inRNase H activity and a β subunit also reduced or substantially reducedin RNase H activity (i.e., AMV αH⁻/βH⁻ RT). In another preferred aspect,the AMV RT may comprise two β subunits, either or both of which may ormay not be reduced or substantially reduced in RNAse H activity (i.e.,AMV βH⁻/βH⁻ RT; AMV βH⁻/βH⁺ RT; or AMV βH⁺βH⁺ RT). In another preferredaspect, the AMV RT may comprise a single α subunit, which may or may notbe reduced or substantially reduced in RNAse H activity (ie., AMV αH⁻ RTor AMV αH⁺ RT). In another preferred aspect, the AMV RT may comprise oneor more βp4 subunits, any or all of which may or may not be reduced orsubstantially reduced in RNAse H activity (e.g., AMV βp4H⁻/βp4H⁻ RT; AMVβp4H⁻/βp4H⁺ RT; AMV βp4H⁺/βp4H⁺ RT; AMV αH⁻/βp4H⁺ RT; AMV αH⁺/βp4H⁺ RT;AMV αH⁻/βp4H⁻ RT; AMV αH⁺/βp4H⁻ RT; AMV βH⁻/βp4H⁻ RT; AMV βH⁺/βp4H⁻ RT;etc.).

In a related aspect of the invention, where the AMV RTs comprise two ormore subunits (or derivatives, variants, fragments or mutants thereof)and preferably comprise two subunits (e.g., a dimer), at least one butpreferably not all of these subunits may be modified or mutated toreduce, substantially reduce or eliminate the polymerase activity of atleast one subunit (e.g., pol−). In a preferred aspect, for an AMV RTwhich comprises an α and β subunit, the β subunit has been modified ormutated (preferably by recombinant techniques) to reduce, substantiallyreduce or eliminate the polymerase activity while the polymeraseactivity of the α subunit has not been mutated or modified in thismanner. Preferably, the α subunit of such AMV RT has also been modifiedor mutated to reduce or substantially reduce RNase H activity while theβ subunit has not been mutated or modified in this manner. Such aconstruct may be designated αH−/βH+pol−. Any number of combinations ofsubunits can be prepared in which mutations or modifications are made inone subunit of a two subunit enzyme, and these constructs may becombined with other modifications or mutations, such as those whichreduce or substantially reduce RNase H activity. Illustrated examplesinclude but are not limited to AMV αH−/βH−pol−; AMV αH+/βH+pol−; AMVαH−pol−/βH+; AMV αH−pol−/βH−; AMV αH+pol−/βH+; AMV βH−/βH−pol−; AMVβH−/βH+pol−; AMV βH+/βH+pol−; AMV βp4H−/βp4H−pol−; AMV βp4H−/βp4H+pol−;AMV βp4H+/βp4H+pol−; AMV αH−/βp4H−pol−; AMV αH+/βp4H+pol−; AMVαH+/βp4H−pol−; AMV βH−/βp4H+pol−; AMV βH−/βp4H+pol−; AMV βH+/βp4H−pol−;etc. In a preferred aspect, the polymerase domain of the subunit ismodified or mutated (one or more point mutations, deletion mutationsand/or insertion mutations) by recombinant techniques. In anotheraspect, the nucleotide binding site of the polymerase domain is modifiedor mutated. In a preferred aspect, one or more acidic amino acids withinthe nucleotide binding site are substituted with different amino acids.Particularly preferred amino acids within the nucleotide binding sitefor mutation or modification include, but are not limited to, Asp¹⁰⁷,Leu¹⁰⁸, Lys¹⁰⁹, and Asp¹¹⁰, or the corresponding amino acid sequence.

The invention also relates to methods of producing the AMV RTs of theinvention, which methods comprise obtaining a host cell comprising anucleic acid sequence encoding one or more α subunits (or derivatives,variants, fragments or mutants thereof) and/or a nucleic acid sequenceencoding one or more β subunits (or derivatives, variants, fragments ormutants thereof) and/or a nucleic acid sequence encoding one or more βp4subunits (or derivatives, variants, fragments or mutants thereof), andculturing the host cell under conditions sufficient to produce the AMVRTs. The nucleic acid sequences encoding such α subunit(s) and/or such βsubunit(s) and/or such βp4 subunit(s) may be contained in the samevector or in different vectors. In accordance with the invention, such αsubunit(s) and/or β subunit(s) and/or βp4 subunit(s) may be producedseparately and mixed before or after isolation of each subunit to formthe AMV RTs of the invention. Alternatively, such a and/or β and/or βp4subunits may be expressed simultaneously (i.e., co-expressed) in thesame host cell, thereby producing an AMV RT comprising an α and a βsubunit, an α subunit alone, a β subunit alone, a βp4 subunit alone, a βsubunit and a βp4 subunit, two β subunits, or two βp4 subunits, orderivatives, variants, fragments or mutants thereof. In a preferredaspect, the α subunit is simultaneously expressed in a host cell withthe β subunit. In a related aspect of the invention, the β or βp4subunits (or derivatives, variants, fragments or mutants thereof) may beexpressed in a host or host cell which accomplishes in vivo processingas described above for production of RSV RTs. Thus, by expressing a βsubunit and/or a βp4 subunit (or derivatives, fragments or mutantsthereof) in a host system which has appropriate processing enzymes orproteins, the corresponding α subunit may be produced allowing formationof an AMV RT which comprises an α and a β subunit, an α and a βp4subunit, etc. As noted above, any or all of the α and/or β and/or βp4subunits (or derivatives, variants, fragments or mutants thereof) of theAMV RT may be reduced or substantially reduced in RNase H activity.These AMV RTs or subunits thereof may then be isolated from the hostcell and may be substantially purified by those methods described abovefor purification of RSV RTs. The invention also relates to kitscomprising one or more of the AMV RTs of the invention.

The invention also relates to other substantially pure ASLV reversetranscriptases, which may or may not be reduced or substantially reducedin RNase H activity. Such ASLV RTs may comprise one or more subunits (orderivatives, variants, fragments or mutants thereof) selected from oneor more α subunits, one or more β subunits, and one or more βp4subunits, as described for RSV RT and AMV RT above. The invention alsorelates to methods of producing such ASLV RTs of the invention asdescribed above for RSV RT and AMV RT.

The invention also concerns the RSV reverse transcriptases and AMVreverse transcriptases or other ASLV reverse transcriptases and subunitsthereof (and derivatives, variants, fragments and mutants thereof of theinvention which have functional activity, as measured by the ability ofthe proteins to produce first strand cDNA from a mRNA template. Suchfunctional activity may be measured in accordance with the inventionbased on the total full-length reverse transcribed product made duringthe synthesis reaction. The amount of product is preferably measuredbased on the mass (e.g., nanograms) of products produced, although othermeans of measuring the amount of product will be recognized by one ofordinary skill in the art. Additionally, functional activity may bemeasured in terms of the percentage of full-length products producedduring a cDNA synthesis reaction. For example, the percent full-lengthfunctional activity may be determined by dividing the amount offull-length product by the amount of total product produced during acDNA synthesis reaction and multiplying the result by 100 to obtain thepercentage. The RSV and AMV reverse transcriptases and their subunits(and derivatives, variants, fragments and mutants thereof) of theinvention produce greater than about 4%, preferably greater than about5%, more preferably greater than about 7.5%, still more preferablygreater than about 10%, still more preferably greater than about 20/,and most preferably greater than about 25%, full-length cDNA in anucleic acid synthesis reaction. Preferred ranges of such percentagesinclude about 5% to about 100%, about 7.5% to about 75%, about 7.5% toabout 50%, about 10% to about 50%, about 15% to about 40%, about 20% toabout 40%, and about 20% to about 50%. Functional activity may also bemeasured in accordance with the invention by determining the percentageof total cDNA product compared to the amount of input mRNA in thesynthesis reaction. Thus, the total amount of cDNA product is divided bythe amount of input mRNA, the result of which is multiplied by 100 todetermine the percentage functional activity associated with amount ofproduct produced compared to amount of the template used. Preferably,the reverse transcriptases of the invention produce greater than about15%, more preferably greater than about 20%, still more preferablygreater than about 25%, still more preferably greater than about 30%,and most preferably greater than about 40%, of cDNA compared to inputmRNA in the cDNA synthesis reaction. Preferred ranges of suchpercentages include about 5% to about 100%, about 10% to about 80%,about 15% to about 80%, about 15% to about 75% about 20% to about 75%,about 20% to about 70%, about 25% to about 75%, about 25% to about 70%,about 25% to about 60%, and about 25% to about 50%. The AMV and RSVreverse transcriptases and their subunits (and derivatives, variants,fragments and mutants thereof) of the invention preferably have specificactivities greater than about 5 units/mg, more preferably greater thanabout 50 units/mg, still more preferably greater than about 100units/mg, 250 units/mg, 500 units/mg, 1000 units/mg, 5000 units/mg or10,000 units/mg, and most preferably greater than about 15,000 units/mg,greater than about 16,000 units/mg, greater than about 17,000 units/mg,greater than about 18,000 units/mg, greater than about 19,000 units/mgand greater than about 20,000 units/mg. Preferred ranges of specificactivities for the AMV and RSV RTs and their subunits (or derivatives,variants, fragments or mutants thereof) of the invention include aspecific activity from about 5 units/mg to about 140,000 units/mg, aspecific activity from about 5 units/mg to about 125,000 units/mg, aspecific activity of from about 50 units/mg to about 100,000 units/mg, aspecific activity from about 100 units/mg to about 100,000 units/mg, aspecific activity from about 250 units/mg to about 100,000 units/mg, aspecific activity from about 500 units/mg to about 100,000 units/mg, aspecific activity from about 1000 units/mg to about 100,000 units/mg, aspecific activity from about 5000 units/mg to about 100,000 units/mg, aspecific activity from about 10,000 units/mg to about 100,000 units/mg,a specific activity from about 25,000 units/mg to about 75,000 units/mg.Other preferred ranges of specific activities include a specificactivity of from about 20,000 units/mg to about 140,000 units/mg, aspecific activity from about 20,000 units/mg to about 130,000 units/mg,a specific activity from about 20,000 units/mg to about 120,000units/mg, a specific activity from about 20,000 units/mg to about110,000 units/mg, a specific activity from about 20,000 units/mg toabout 100,000 units/mg, a specific activity from about 20,000 units/mgto about 90,000 units/mg, a specific activity from about 25,000 units/mgto about 140,000 units/mg, a specific activity from about 25,000units/mg to about 130,000 units/mg, a specific activity from about25,000 units/mg to about 120,000 units/mg, a specific activity fromabout 25,000 units/mg to about 110,000 units/mg, a specific activityfrom about 25,000 units/mg to about 100,000 units/mg, and a specificactivity from about 25,000 units/mg to about 90,000 units/mg.Preferably, the lower end of the specific activity range may vary from30,000, 35,000, 40,000, 45,000, 50,000, 5,000, 60,000, 65,000, 70,000,75,000, and 80,000 units/mg, while the upper end of the range may varyfrom 150,000, 140,000, 130,000, 120,000, 110,000, 100,000, and 90,000units/mg. In accordance with the invention, specific activity is ameasurement of the enzymatic activity (in units) of the protein orenzyme relative to the total amount of protein or enzyme used in areaction. The measurement of a specific activity may be determined bystandard techniques well-known to one of ordinary skill in the art.Preferred assays for determining the specific activity of an enzyme orprotein are described in detail in the Examples below.

The RSV RTs and AMV RTs or other ASLV RTs and their subunits (orderivatives, variants, fragments or mutants thereof) of the inventionmay be used to make nucleic acid molecules from one or more templates.Such methods comprise mixing one or more nucleic acid templates (e.g.,mRNA, and more preferably a population of mRNA molecules) with one ormore of the RSV RTs and/or one or more AMV RTs and/or other ASLV RTs ofthe invention and incubating the mixture under conditions sufficient tomake one or more nucleic acid molecules complementary to all or aportion of the one or more nucleic acid templates.

The invention also relates to methods for the amplification of one ormore nucleic acid molecules comprising mixing one or more nucleic acidtemplates with one or more of the RSV RTs and/or one or more of the AMVRTs and/or other ASLV RTs of the invention and optionally with one ormore DNA polymerases, and incubating the mixture under conditionssufficient to amplify one or more nucleic acid molecules complementaryto all or a portion of the one or more nucleic acid templates.

The invention also concerns methods for the sequencing of one or morenucleic acid molecules comprising (a) mixing one or more nucleic acidmolecules to be sequenced with one or more primer nucleic acidmolecules, one or more RSV RTs and/or one or more AMV RTs and/or otherASLV RTs of the invention, one or more nucleotides and one or moreterminating agents; (b) incubating the mixture under conditionssufficient to synthesize a population of nucleic acid moleculescomplementary to all or a portion of the one or more nucleic acidmolecules to be sequenced; and (c) separating the population of nucleicacid molecules to determine the nucleotide sequence of all or a portionof the one or more nucleic acid molecules to be sequenced.

The invention also concerns methods for elevated- or high-temperaturereverse transcription of a nucleic acid molecule comprising (a) mixing anucleic acid template (preferably an RNA (e.g., a mRNA molecule or apolyA+RNA molecule) or a DNA molecule) with one or more polypeptideshaving reverse transcriptase activity; and (b) incubating the mixture ata temperature of 50° C. or greater and under conditions sufficient tomake a first nucleic acid molecule (such as a full length cDNA molecule)complementary to all or a portion of the nucleic acid template. Inpreferred such methods, the mixture is incubated at a temperature ofabout 51° C. or greater, about 52° C. or greater, about 53° C. orgreater, about 54° C. or greater, about 55° C. or greater, about 56° C.or greater, about 57° C. or greater, about 58° C. or greater, about 59°C. or greater, about 60° C. or greater, about 61° C. or greater, about62° C. or greater; or at a temperature range of from about 50° C. toabout 70° C., about 51° C. to about 70° C., about 52° C. to about 70°C., about 53° C. to about 70° C., about 54° C. to about 70° C., about55° C. to about 70° C., about 55° C. to about 65° C., about 56° C. toabout 65° C., about 56° C. to about 64° C. or about 56° C. to about 62°C. The invention is also directed to such methods which further compriseincubating the first nucleic acid molecule under conditions sufficientto make a second nucleic acid molecule complementary to all or portionof the first nucleic acid molecule. According to the invention, thefirst and second nucleic acid molecules produced by these methods may beDNA molecules, and may form a double stranded DNA molecule which may bea full length cDNA molecule. The one or more polypeptides having reversetranscriptase activity that are used in these methods preferably arereduced or substantially reduced in RNase H activity, and may beselected from the group consisting of one or more subunits of AMVreverse transcriptase and one or more subunits of RSV reversetranscriptase and one or more subunits of other ASLV reversetranscriptases (or derivatives, variants, fragments or mutants thereof).As noted above, such AMV RTs or RSV RTs or other ASLV RTs may compriseone or more α subunits, one or more β subunits, and/or one or more βp4subunits, any or all of which subunits may be reduced or substantiallyreduced in RNase H activity. Particularly preferred polymerases havingRT activity for use in these methods are those RSV RTs and AMV RTs orother ASLV RTs and their subunits (or derivatives, variants, fragmentsor mutants thereof) described above.

The invention also concerns nucleic acid molecules produced by suchmethods (which may be full-length cDNA molecules), vectors (particularlyexpression vectors) comprising these nucleic acid molecules and hostcells comprising these vectors and nucleic acid molecules.

Sources of Enzymes

Enzymes for use in the compositions, methods and kits of the inventioninclude any enzyme having reverse transcriptase activity. Such enzymesinclude, but are not limited to, retroviral reverse transcriptase,retrotransposon reverse transcriptase, hepatitis B reversetranscriptase, cauliflower mosaic virus reverse transcriptase, bacterialreverse transcriptase, Tth DNA polymerase, Taq DNA polymerase (Saiki, R.K., et al., Science 239:487-491 (1988); U.S. Pat. Nos. 4,889,818 and4,965,188), Tne DNA polymerase (WO 96/10640), Tma DNA polymerase (U.S.Pat. No. 5,374,553) and mutants, fragments, variants or derivativesthereof (see, e.g., commonly owned, co-pending U.S. patent applicationSer. Nos. 08/706,702 and 08/706,706, both filed Sep. 9, 1996, which areincorporated by reference herein in their entireties). As will beunderstood by one of ordinary skill in the art, modified reversetranscriptases may be obtained by recombinant or genetic engineeringtechniques that are routine and well-known in the art. Mutant reversetranscriptases can, for example, be obtained by mutating the gene orgenes encoding the reverse transcriptase of interest by site-directed orrandom mutagenesis. Such mutations may include point mutations, deletionmutations and insertional mutations. Preferably, one or more pointmutations (e.g., substitution of one or more amino acids with one ormore different amino acids) are used to construct mutant reversetranscriptases of the invention. Fragments of reverse transcriptases maybe obtained by deletion mutation by recombinant techniques that areroutine and well-known in the art, or by enzymatic digestion of thereverse transcriptase(s) of interest using any of a number of well-knownproteolytic enzymes.

Preferred enzymes for use in the invention include those that arereduced or substantially reduced in RNase H activity. Such enzymes thatare reduced or substantially reduced in RNase H activity may be obtainedby mutating the RNase H domain within the reverse transcriptase ofinterest, preferably by one or more point mutations, one or moredeletion mutations, and/or one or more insertion mutations as describedabove. By an enzyme “substantially reduced in RNase H activity” is meantthat the enzyme has less than about 30%, less than about 25%, 20%, morepreferably less than about 15%, less than about 10%, less than about7.5%, or less than about 5%, and most preferably less than about 5% orless than about 2%, of the RNase H activity of the correspondingwildtype or RNase H⁺ enzyme such as wildtype Moloney Murine LeukemiaVirus (M-MLV), Avian Myeloblastosis Virus (AMV) or Rous Sarcoma Virus(RSV) reverse transcriptases. The RNase H activity of any enzyme may bedetermined by a variety of assays, such as those described, for example,in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988), in Gerard, G. F., et al., FOCUS 14(5):91 (1992), and inU.S. Pat. No. 5,668,005, the disclosures of all of which are fullyincorporated herein by reference.

Particularly preferred enzymes for use in the invention include, but arenot limited to, M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV H⁻ reversetranscriptase, MAV H⁻ reverse transcriptase and HIV H⁺ reversetranscriptase. It will be understood by one of ordinary skill, however,that any enzyme capable of producing a DNA molecule from a ribonucleicacid molecule (i.e., having reverse transcriptase activity) that issubstantially reduced in RNase H activity may be equivalently used inthe compositions, methods and kits of the invention.

Enzymes used in the invention may have distinct reverse transcriptionpause sites with respect to the template nucleic acid. Whether or nottwo enzymes have distinct reverse transcription pause sites may bedetermined by a variety of assays, including, for example,electrophoretic analysis of the chain lengths of DNA molecules producedby the two enzymes (Weaver, D. T., and DePamphilis, M. L., J. Biol.Chem. 257(4):2075-2086 (1982); Abbots, J., et al., J. Biol. Chem.268(14):10312-10323 (1993)), or by other assays that will be familiar toone of ordinary skill in the art. As described above, these distincttranscription pause sites may represent secondary structural andsequence barriers in the nucleic acid template which occur frequently athomopolymer stretches. Thus, for example, the second enzyme may reversetranscribe to a point (e.g., a hairpin) on the template nucleic acidthat is proximal or distal (i.e., 3′ or 5′) to the point to which thefirst enzyme reverse transcribes the template nucleic acid. Thiscombination of two or more enzymes having distinct reverse transcriptionpause sites facilitates production of full-length cDNA molecules sincethe secondary structural and sequence barriers may be overcome.Moreover, the elevated- or high-temperature reverse transcription of theinvention may also assist in overcoming secondary structural andsequence barriers during nucleic acid synthesis. Thus, the elevated- orhigh-temperature synthesis may be used in combination with the two ormore reverse transcriptases (preferably using an AMV RT, an RSV RT orother ASLV RT) to facilitate full-length cDNA synthesis.

A variety of DNA polymerases are useful in accordance with the presentinvention. Such polymerases include, but are not limited to, Thermusthermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neapolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Pyrococcus furiosis (Pfu) DNA polymerase, DEEPVENT™ DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillussterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNApolymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasmaacidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNApolymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, Mycobacterium spp. DNA polymerase (Mtb, Mlep), andmutants, variants and derivatives thereof.

DNA polymerases used in accordance with the invention may be any enzymethat can synthesize a DNA molecule from a nucleic acid template, itstypically in the 5′ to 3′ direction. Such polymerases may be mesophilicor thermophilic, but are preferably thermophilic. Mesophilic polymerasesinclude T5 DNA polymerase, T7 DNA polymerase, Klenow fragment DNApolymerase, DNA polymerase III, and the like. Preferred DNA polymerasesare thermostable DNA polymerases such as Taq, Tne, Tma, Pfu, VENT™,DEEPVENT™, Tth and mutants, variants and derivatives thereof (U.S. Pat.Nos. 5,436,149; 5,512,462; WO 92/06188; WO 92/06200; WO 96/10640;Barnes, W. M., Gene 112:29-35 (1992), Lawyer, F. C., et al., PCR Meth.Appl. 2:275-287 (1993); Flaman, J.-M., et al., Nucl. Acids Res.22(15):3259-3260 (1994)). For amplification of long nucleic acidmolecules (e.g., nucleic acid molecules longer than about 3-5 Kb inlength), at least two DNA polymerases (one substantially lacking 3′exonuclease activity and the other having 3′ exonuclease activity) aretypically used. See U.S. Pat. Nos. 5,436,149; 5,512,462; Barnes, W. M.,Gene 112:29-35 (1992); and commonly owned, co-pending U.S. patentapplication Ser. No.08/801,720, filed Feb. 14, 1997, the disclosures ofall of which are incorporated herein in their entireties. Examples ofDNA polymerases substantially lacking in 3′ exonuclease activityinclude, but are not limited to, Taq, Tne(exo⁻), Tma, Pfu(exo⁻), Pwo andTth DNA polymerases, and mutants, variants and derivatives thereof.Nonlimiting examples of DNA polymerases having 3′ exonuclease activityinclude Pfu/DEEPVENT™ and Tli/VENT™ and mutants, variants andderivatives thereof.

Polypeptides having reverse transcriptase activity for use in theinvention may be obtained commercially, for example from LifeTechnologies, Inc. (Rockville, Md.), Pharmacia (Piscataway, N.J.), Sigma(Saint Louis, Mo.) or Boehringer Mannheim Biochemicals (Indianapolis,Ind.). Alternatively, polypeptides having reverse transcriptase activitymay be isolated from their natural viral or bacterial sources accordingto standard procedures for isolating and purifying natural proteins thatare well-known to one of ordinary skill in the art (see, e.g., Houts, G.E., et al., J. Virol. 29:517(1979)). In addition, the polypeptideshaving reverse transcriptase activity may be prepared by recombinant DNAtechniques that are familiar to one of ordinary skill in the art (see,e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis,D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376(1988)).

DNA polymerases for use in the invention may be obtained commercially,for example from Life Technologies, Inc. (Rockville, Md.), Perkin-Elmer(Branchburg, N.J.), New England BioLabs (Beverly, Mass.) or BoehringerMannheim Biochemicals (Indianapolis, Ind.).

Formulation of Enzyme Compositions

To form the compositions of the present invention, two or more reversetranscriptases are preferably admixed in a buffered salt solution. Oneor more DNA polymerases and/or one or more nucleotides may optionally beadded to make the compositions of the invention. More preferably, theenzymes are provided at working concentrations in stable buffered saltsolutions. The terms “stable” and “stability” as used herein generallymean the retention by a composition, such as an enzyme composition, ofat least 70%, preferably at least 80%, and most preferably at least 90%,of the original enzymatic activity (in units) after the enzyme orcomposition containing the enzyme has been stored for about one week ata temperature of about 4° C., about two to six months at a temperatureof about −20° C., and about six months or longer at a temperature ofabout −80° C. As used herein, the term “working concentration” means theconcentration of an enzyme that is at or near the optimal concentrationused in a solution to perform a particular function (such as reversetranscription of nucleic acids).

The water used in forming the compositions of the present invention ispreferably distilled, deionized and sterile filtered (through a 0.1-0.2micrometer filter), and is free of contamination by DNase and RNaseenzymes. Such water is available commercially, for example from SigmaChemical Company (Saint Louis, Mo.), or may be made as needed accordingto methods well known to those skilled in the art.

In addition to the enzyme components, the present compositionspreferably comprise one or more buffers and cofactors necessary forsynthesis of a nucleic acid molecule such as a cDNA molecule.Particularly preferred buffers for use in forming the presentcompositions are the acetate, sulfate, hydrochloride, phosphate or freeacid forms of Tris-(hydroxymethyl)aminomethane (TRIS®), althoughalternative buffers of the same approximate ionic strength and pKa asTRIS® may be used with equivalent results. In addition to the buffersalts, cofactor salts such as those of potassium (preferably potassiumchloride or potassium acetate) and magnesium (preferably magnesiumchloride or magnesium acetate) are included in the compositions.Addition of one or more carbohydrates and/or sugars to the compositionsand/or synthesis reaction mixtures may also be advantageous, to supportenhanced stability of the compositions and/or reaction mixtures uponstorage. Preferred such carbohydrates or sugars for inclusion in thecompositions and/or synthesis reaction mixtures of the inventioninclude, but are not limited to, sucrose, trehalose, and the like.Furthermore, such carbohydrates and/or sugars may be added to thestorage buffers for the enzymes used in the production of the enzymecompositions and kits of the invention. Such carbohydrates and/or sugarsare commercially available from a number of sources, including Sigma(St. Louis, Mo.).

It is often preferable to first dissolve the buffer salts, cofactorsalts and carbohydrates or sugars at working concentrations in water andto adjust the pH of the solution prior to addition of the enzymes. Inthis way, the pH-sensitive enzymes will be less subject to acid- oralkaline-mediated inactivation during formulation of the presentcompositions.

To formulate the buffered salts solution, a buffer salt which ispreferably a salt of Tris(hydroxymethyl)aminomethane (TRIS®), and mostpreferably the hydrochloride salt thereof, is combined with a sufficientquantity of water to yield a solution having a TRIS® concentration of5-150 millimolar, preferably 10-60 millimolar, and most preferably about20-60 millimolar. To this solution, a salt of magnesium (preferablyeither the chloride or acetate salt thereof may be added to provide aworking concentration thereof of 1-10 millimolar, preferably 1.5-8.0millimolar, and most preferably about 3-7.5 millimolar. A salt ofpotassium (preferably a chloride or acetate salt of potassium) may alsobe added to the solution, at a working concentration of 10-100millimolar and most preferably about 75 millimolar. A reducing agentsuch as dithiothreitol may be added to the solution, preferably at afinal concentration of about 1-100 mM, more preferably a concentrationof about 5-50 mM or about 7.5-20 mM, and most preferably at aconcentration of about 10 mM. Preferred concentrations of carbohydratesand/or sugars for inclusion in the compositions of the invention rangefrom about 5% (w/v) to about 30% (w/v), about 7.5% (w/v) to about 25%(w/v), about 10% (w/v) to about 25% (w/v), about 10% (w/v) to about 20%(w/v), and preferably about 10% (w/v) to about 15% (w/v). A small amountof a salt of ethylenediaminetetraacetate (EDTA), such as disodium EDTA,may also be added (preferably about 0.1 millimolar), although inclusionof EDTA does not appear to be essential to the function or stability ofthe compositions of the present invention. After addition of all buffersand salts, this buffered salt solution is mixed well until all salts aredissolved, and the pH is adjusted using methods known in the art to a pHvalue of 7.4 to 9.2, preferably 8.0 to 9.0, and most preferably about8.4.

To these buffered salt solutions, the enzymes (reverse transcriptasesand/or DNA polymerases) are added to produce the compositions of thepresent invention. M-MLV RTs are preferably added at a workingconcentration in the solution of about 1,000 to about 50,000 units permilliliter, about 2,000 to about 30,000 units per milliliter, about2,500 to about 25,000 units per milliliter, about 3,000 to about 22,500units per milliliter, about 4,000 to about 20,000 units per milliliter,and most preferably at a working concentration of about 5,000 to about20,000 units per milliliter. AMV RTs, MAV RTs, RSV RTs and RAV RTs,including those of the invention described above, are preferably addedat a working concentration in the solution of about 100 to about 5000units per milliliter, about 125 to about 4000 units per milliliter,about 150 to about 3000 units per milliliter, about 200 to about 2500units per milliliter, about 225 to about 2000 units per milliliter, andmost preferably at a working concentration of about 250 to about 1000units per milliliter. The enzymes in the thermophilic DNA polymerasegroup (Taq, Tne, Tma, Pfu, VENT™, DEEPVENT™, Tth and mutants, variantsand derivatives thereof) are preferably added at a working concentrationin the solution of about 100 to about 1000 units per milliliter, about125 to about 750 units per milliliter, about 150 to about 700 units permilliliter, about 200 to about 650 units per milliliter, about 225 toabout 550 units per milliliter, and most preferably at a workingconcentration of about 250 to about 500 units per milliliter. Theenzymes may be added to the solution in any order, or may be addedsimultaneously.

The compositions of the invention may further comprise one or morenucleotides, which are preferably deoxynucleoside triphosphates (dNTPs)or dideoxynucleoside triphosphates (ddNTPs). The dNTP components of thepresent compositions serve as the “building blocks” for newlysynthesized nucleic acids, being incorporated therein by the action ofthe polymerases, and the ddNTPs may be used in sequencing methodsaccording to the invention. Examples of nucleotides suitable for use inthe present compositions include, but are not limited to, dUTP, dATP,dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, α-thio-dATP, α-thio-dTTP,α-thio-dGTP, α-thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, ddGTP, ddITP,7-deaza-ddGTP, α-thio-ddATP, α-thio-ddTTP, α-thio-ddGTP, α-thio-ddCTP orderivatives thereof, all of which are available commercially fromsources including Life Technologies, Inc. (Rockville, Md.), New EnglandBioLabs (Beverly, Mass.) and Sigma Chemical Company (Saint Louis, Mo.).The nucleotides may be unlabeled, or they may be detectably labeled bycoupling them by methods known in the art with radioisotopes (e.g., ³H,¹⁴C, ³²P or ³⁵S), vitamins (e.g., biotin), fluorescent moieties (e.g.,fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescentlabels (e.g., using the PHOTO-GENE™ or ACES™ chemiluminescence systems,available commercially from Life Technologies, Inc., Rockville, Md.),dioxigenin and the like. Labeled nucleotides may also be obtainedcommercially, for example from Life Technologies, Inc. (Rockville, Md.)or Sigma Chemical Company (Saint Louis, Mo.). In the presentcompositions, the nucleotides are added to give a working concentrationof each nucleotide of about 10-4000 micromolar, about 50-2000micromolar, about 100-1500 micromolar, or about 200-1200 micromolar, andmost preferably a concentration of about 1000 micromolar.

To reduce component deterioration, storage of the reagent compositionsis preferably at about 4° C. for up to one day, or most preferably at−20° C. for up to one year.

In another aspect, the compositions and reverse transcriptases of theinvention may be prepared and stored in dry form in the presence of oneor more carbohydrates, sugars, or synthetic polymers. Preferredcarbohydrates, sugars or polymers for the preparation of driedcompositions or reverse transcriptases include, but are not limited to,sucrose, trehalose, and polyvinylpyrrolidone (PVP) or combinationsthereof. See, e.g., U.S. Pat. Nos. 5,098,893, 4,891,319, and 5,556,771,the disclosures of which are entirely incorporated herein by reference.Such dried compositions and enzymes may be stored at varioustemperatures for extended times without significant deterioration ofenzymes or components of the compositions of the invention. Preferably,the dried reverse transcriptases or compositions are stored at 4° C. orat −20° C.

Production of cDNA Molecules

Sources of Nucleic Acid Molecules

In accordance with the invention, cDNA molecules (single-stranded ordouble-stranded) may be prepared from a variety of nucleic acid templatemolecules. Preferred nucleic acid molecules for use in the presentinvention include single-stranded or double-stranded DNA and RNAmolecules, as well as double-stranded DNA:RNA hybrids. More preferrednucleic acid molecules include messenger RNA (mRNA), transfer RNA (tRNA)and ribosomal RNA (rRNA) molecules, although mRNA molecules are thepreferred template according to the invention.

The nucleic acid molecules that are used to prepare cDNA moleculesaccording to the methods of the present invention may be preparedsynthetically according to standard organic chemical synthesis methodsthat will be familiar to one of ordinary skill. More preferably, thenucleic acid molecules may be obtained from natural sources, such as avariety of cells, tissues, organs or organisms. Cells that may be usedas sources of nucleic acid molecules may be prokaryotic (bacterialcells, including but not limited to those of species of the generaEscherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Xanthomonas and Streptomyces) or eukaryotic(including fungi (especially yeasts), plants, protozoans and otherparasites, and animals including insects (particularly Drosophila spp.cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly human cells)).

Mammalian somatic cells that may be used as sources of nucleic acidsinclude blood cells (reticulocytes and leukocytes), endothelial cells,epithelial cells, neuronal cells (from the central or peripheral nervoussystems), muscle cells (including myocytes and myoblasts from skeletal,smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes)may also be used as sources of nucleic acids for use in the invention,as may the progenitors, precursors and stem cells that give rise to theabove somatic and germ cells. Also suitable for use as nucleic acidsources are mammalian tissues or organs such as those derived frombrain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous,skin, genitourinary, circulatory, lymphoid, gastrointestinal andconnective tissue sources, as well as those derived from a mammalian(including human) embryo or fetus.

Any of the above prokaryotic or eukaryotic cells, tissues and organs maybe normal, diseased, transformed, established, progenitors, precursors,fetal or embryonic. Diseased cells may, for example, include thoseinvolved in infectious diseases (caused by bacteria, fungi or yeast,viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) orparasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, muscular dystrophy ormultiple sclerosis) or in cancerous processes. Transformed orestablished animal cell lines may include, for example, COS cells, CHOcells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293cells, L929 cells, F9 cells, and the like. Other cells, cell lines,tissues, organs and organisms suitable as sources of nucleic acids foruse in the present invention will be apparent to one of ordinary skillin the art.

Once the starting cells, tissues, organs or other samples are obtained,nucleic acid molecules (such as mRNA) may be isolated therefrom bymethods that are well-known in the art (See, e.g., Maniatis, T., et al.,Cell 15:687-701 (1978); Okayama, H., and Berg, P., Mol. Cell. Biol.2:161-170 (1982); Gubler, U., and Hoffman, B. J., Gene 25:263-269(1983)). The nucleic acid molecules thus isolated may then be used toprepare cDNA molecules and cDNA libraries in accordance with the presentinvention.

In the practice of the invention, cDNA molecules or cDNA libraries areproduced by mixing one or more nucleic acid molecules obtained asdescribed above, which is preferably one or more mRNA molecules such asa population of mRNA molecules, with two or more polypeptides havingreverse transcriptase activity, or with one or more of the compositionsof the invention or with one or more of the RSV RTs and/or AMV RTsand/or other ASLV RTs of the invention, under conditions favoring thereverse transcription of the nucleic acid molecule by the action of theenzymes or the compositions to form a cDNA molecule (single-stranded ordouble-stranded). Thus, the method of the invention comprises (a) mixingone or more nucleic acid templates (preferably one or more RNA or mRNAtemplates, such as a population of mRNA molecules) with one or morereverse transcriptases of the invention and (b) incubating the mixtureunder conditions sufficient to make one or more nucleic acid moleculescomplementary to all or a portion of the one or more templates. Suchmethods may include the use of one or more DNA polymerases. Theinvention may be used in conjunction with methods of cDNA synthesis suchas those described in the Examples below, or others that are well-knownin the art (see, e.g., Gubler, U., and Hoffinan, B. J., Gene25:263-269(1983); Krug, M. S., and Berger, S. L.,Meth. Enzymo.152:316-325 (1987); Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, pp. 8.60-8.63 (1989)), to produce cDNA molecules orlibraries.

The invention is also particularly directed to methods for reversetranscription of a nucleic acid molecule at elevated temperatures. Asdescribed in more detail in Example 5, retroviral RTs are generally notused at temperatures above 37° C. to 42° C. to copy nucleic acidtemplates such as RNA molecules because of the limited thermal stabilityof these mesophilic enzymes. At these temperatures, however, mRNAsecondary structure may interfere with reverse transcription (Gerard, G.F., et al., FOCUS 11:60 (1989), Myers, T. W., and Gelfand, D. H.,Biochem. 30:7661 (1991)), and the specificity of primer binding may bereduced during gene-specific reverse transcription processes, such asRT-PCR, causing high background signal (Myers, T. W., and Gelfand, D.H., Biochem. 30:7661 (1991); Freeman, W. N., et al., BioTechniques20:782 (1996)). To help overcome these problems, the present inventiontherefore provides methods of RNA reverse transcription at more elevatedtemperatures, i.e., above 50° C.

Therefore, the invention is related to methods for reverse transcriptionof a nucleic acid molecule comprising (a) mixing a nucleic acid templatewith one or more polypeptides having reverse transcriptase activity; and(b) incubating the mixture at a temperature of about 50° C. or greaterand under conditions sufficient to make a first nucleic acid moleculecomplementary to all or a portion of the nucleic acid template. Nucleicacid templates which may be copied according to these methods include,but are not limited to, an RNA molecule (e.g., a mRNA molecule or apolyA+ RNA molecule) and a DNA molecule (e.g., a single-stranded ordouble-stranded DNA molecule). According to the invention, the firstnucleic acid molecule produced by these methods may be a full lengthcDNA molecule. While any incubation temperature of about 50° C. orgreater may be used in the present methods, particularly preferredincubation temperatures include, but are not limited to, temperatures ofabout 51° C. or greater, about 52° C. or greater, about 53° C. orgreater, about 54° C. or greater, about 55° C. or greater, about 56° C.or greater, about 57° C. or greater, about 58° C. or greater, about 59°C. or greater, about 60° C. or greater, about 61° C. or greater, about62° C. or greater, about 63° C. or greater, about 64° C. or greater,about 65° C. or greater, about 66° C. or greater, about 67° C. orgreater, about 68° C. or greater, about 69° C. or greater or about 70°C. or greater. In other such methods, the incubation temperature may beover a range of incubation temperatures, including but not limited to atemperature range of from about 50° C. to about 70° C., about 51° C. toabout 70° C., about 52° C. to about 70° C., about 53° C. to about 70°C., about 54° C. to about 70° C., about 55° C. to about 70° C., about55° C. to about 69° C., about 55° C. to about 68° C., about 55° C. toabout 67° C., about 55° C. to about 66° C., about 55° C. to about 65°C., about 56° C. to about 65° C., about 56° C. to about 64° C. or about56° C. to about 62° C. The invention is also directed to such methodswhich further comprise incubating the first nucleic acid molecule underconditions sufficient to make a second nucleic acid moleculecomplementary to all or portion of the first nucleic acid molecule.According to the invention, the first and second nucleic acid moleculesproduced by these methods may be DNA molecules, and may form a doublestranded DNA molecule which may be a full length cDNA molecule. Asdescribed for the methods above, the one or more polypeptides havingreverse transcriptase activity that are used in these higher-temperaturemethods preferably are reduced or substantially reduced in RNase Hactivity, and may be selected from the group consisting of one or moreAMV reverse transcriptases or subunits thereof (or derivatives,variants, fragments or mutants thereof), and one or more RSV reversetranscriptases or subunits thereof (or derivatives, variants, fragmentsor mutants thereof) or other ASLV RTs or subunits thereof (orderivatives, variants, fragments or mutants thereof). Particularlypreferred AMV RTs and RSV RTs and other ASLV RTs include those providedby the present invention and described in detail above. Moreparticularly preferred are those AMV RTs and RSV RTs having the genotypeAMV αH⁻/βH⁺ RT or RSV αH⁻/βH⁺ RT. Such constructs are preferably made bymutating or modifying the gene encoding the α subunit to reduce orsubstantially reduce RNase H activity while the gene encoding the βsubunit is not so mutated or modified. The resulting polypeptides(produced by co-expression) will be reduced or substantially reduced inRNase H activity.

Other methods of cDNA synthesis which may advantageously use the presentinvention will be readily apparent to one of ordinary skill in the art.

Having obtained cDNA molecules or libraries according to the presentmethods, these cDNAs may be isolated for further analysis ormanipulation. Detailed methodologies for purification of cDNAs aretaught in the GENETRAPPER™ manual (Life Technologies, Inc.; Rockville,Md.), which is incorporated herein by reference in its entirety,although alternative standard techniques of cDNA isolation such as thosedescribed in the Examples below or others that are known in the art(see, e.g., Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, pp. 8.60-8.63 (1989)) may also be used.

In other aspects of the invention, the invention may be used in methodsfor amplifying and sequencing nucleic acid molecules. Nucleic acidamplification methods according to this aspect of the invention may beone-step (e.g., one-step RT-PCR) or two-step (e.g., two-step RT-PCR)reactions. According to the invention, one-step RT-PCR type reactionsmay be accomplished in one tube thereby lowering the possibility ofcontamination. Such one-step reactions comprise (a) mixing a nucleicacid template (e.g., mRNA) with two or more polypeptides having reversetranscriptase activity and with one or more DNA polymerases and (b)incubating the mixture under conditions sufficient to amplify a nucleicacid molecule complementary to all or a portion of the template.Alternatively, amplification may be accomplished by mixing a templatewith two or more polypeptides having reverse transcriptase activity (andoptionally having DNA polymerase activity). Incubating such a reactionmixture under appropriate conditions allows amplification of a nucleicacid molecule complementary to all or a portion of the template. Suchamplification may be accomplished by the reverse transcriptase activityalone or in combination with the DNA polymerase activity. Two-stepRT-PCR reactions maybe accomplished in two separate steps. Such a methodcomprises (a) mixing a nucleic acid template (e.g., mRNA) with two ormore reverse transcriptases, (b) incubating the mixture under conditionssufficient to make a nucleic acid molecule (e.g., a DNA molecule)complementary to all or a portion of the template, (c) mixing thenucleic acid molecule with one or more DNA polymerases and (d)incubating the mixture of step (c) under conditions sufficient toamplify the nucleic acid molecule. For amplification of long nucleicacid molecules (i.e., greater than about 3-5 Kb in length), acombination of DNA polymerases may be used, such as one DNA polymerasehaving 3′ exonuclease activity and another DNA polymerase beingsubstantially reduced in 3′ exonuclease activity. An alternativetwo-step procedure comprises the use of two or more polypeptides havingreverse transcriptase activity and DNA polymerase activity (e.g., Tth,Tma or Tne DNA polymerases and the like) rather than separate additionof a reverse transcriptase and a DNA polymerase.

Nucleic acid sequencing methods according to this aspect of theinvention may comprise both cycle sequencing (sequencing in combinationwith amplification) and standard sequencing reactions. The sequencingmethod of the invention thus comprises (a) mixing a nucleic acidmolecule to be sequenced with one or more primers, two or more reversetranscriptases, one or more nucleotides and one or more terminatingagents, (b) incubating the mixture under conditions sufficient tosynthesize a population of nucleic acid molecules complementary to allor a portion of the molecule to be sequenced, and (c) separating thepopulation to determine the nucleotide sequence of all or a portion ofthe molecule to be sequenced. According to the invention, one or moreDNA polymerases (preferably thermostable DNA polymerases) may be used incombination with or separate from the reverse transcriptases.

Amplification methods which may be used in accordance with the presentinvention include PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202), StrandDisplacement Amplification (SDA; U.S. Pat. No. 5,455,166; EP 0 684 315),and Nucleic Acid Sequence-Based Amplification (NASBA; U.S. Pat. No.5,409,818; EP 0 329 822). Nucleic acid sequencing techniques which mayemploy the present compositions include dideoxy sequencing methods suchas those disclosed in U.S. Pat. Nos. 4,962,022 and 5,498,523, as well asmore complex PCR-based nucleic acid fingerprinting techniques such asRandom Amplified Polymorphic DNA (RAPD) analysis (Williams, J. G. K., etal., Nucl. Acids Res. 18(22):6531-6535, 1990), Arbitrarily Primed PCR(AP-PCR; Welsh, J., and McClelland, M., Nucl. Acids Res.18(24):7213-7218, 1990), DNA Amplification Fingerprinting (DAF;Caetano-Anollés et al., Bio/Technology 9:553-557, 1991), microsatellitePCR or Directed Amplification of Minisatellite-region DNA (DAMD; Heath,D. D., et al., Nucl. Acids Res. 21(24): 5782-5785, 1993), andAmplification Fragment Length Polymorphism (AFLP) analysis (EP 0 534858; Vos, P., et al., Nucl. Acids Res. 23(21):4407-4414, 1995; Lin, J.J., and Kuo, J., FOCUS 17(2):66-70, 1995). In a particularly preferredaspects, the invention may be used in methods of amplifying orsequencing a nucleic acid molecule comprising one or more polymerasechain reactions (PCRs), such as any of the PCR-based methods describedabove.

Kits

In another embodiment, the present invention may be assembled into kitsfor use in reverse transcription or amplification of a nucleic acidmolecule, or into kits for use in sequencing of a nucleic acid molecule.Kits according to this aspect of the invention comprise a carrier means,such as a box, carton, tube or the like, having in close confinementtherein one or more container means, such as vials, tubes, ampules,bottles and the like, wherein a first container means contains one ormore polypeptides having reverse transcriptase activity. Thesepolypeptides having reverse transcriptase activity may be in a singlecontainer as mixtures of two or more potypeptides, or in separatecontainers, The kits of the invention may also comprise (in the same orseparate containers) one or more DNA polymerases, a suitable buffer, oneor more nucleotides and/or one or more primers.

In a specific aspect of the invention, the reverse transcription andamplification kits may comprise one or more components (in mixtures orseparately) including one or more, preferably two or more, polypeptideshaving reverse transcriptase activity of the invention, one or morenucleotides needed for synthesis of a nucleic acid molecule, and/or aprimer (e.g., oligo(dT) for reverse transcription). Such reversetranscription and amplification kits may further comprise one or moreDNA polymerases. Sequencing kits of the invention may comprise one ormore, preferably two or more, polypeptides having reverse transcriptaseactivity of the invention, and optionally one or more DNA polymerases;one or more terminating agents (e.g., dideoxynucleoside triphosphatemolecules) needed for sequencing of a nucleic acid molecule, one or morenucleotides and/or one or more primers. Preferred polypeptides havingreverse transcriptase activity, DNA polymerases, nucleotides, primersand other components suitable for use in the reverse transcription,amplification and sequencing kits of the invention include thosedescribed above. The kits encompassed by this aspect of the presentinvention may further comprise additional reagents and compoundsnecessary for carrying out standard nucleic acid reverse transcription,amplification or sequencing protocols. Such polypeptides having reversetranscriptase activity of the invention, DNA polymerases, nucleotides,primers, and additional reagents, components or compounds may becontained in one or more containers, and may be contained in suchcontainers in a mixture of two or more of the above-noted components ormay be contained in the kits of the invention in separate containers.

Use of Nucleic Acid Molecules

The nucleic acid molecules or cDNA libraries prepared by the methods ofthe present invention may be further characterized, for example bycloning and sequencing (i.e., determining the nucleotide sequence of thenucleic acid molecule), by the sequencing methods of the invention or byothers that are standard in the art (see, e.g., U.S. Pat. Nos. 4,962,022and 5,498,523, which are directed to methods of DNA sequencing).Alternatively, these nucleic acid molecules may be used for themanufacture of various materials in industrial processes, such ashybridization probes by methods that are well-known in the art.Production of hybridization probes from cDNAs will, for example, providethe ability for those in the medical field to examine a patient's cellsor tissues for the presence of a particular genetic marker such as amarker of cancer, of an infectious or genetic disease, or a marker ofembryonic development. Furthermore, such hybridization probes can beused to isolate DNA fragments from genomic DNA or cDNA librariesprepared from a different cell, tissue or organism for furthercharacterization.

The nucleic acid molecules of the present invention may also be used toprepare compositions for use in recombinant DNA methodologies.Accordingly, the present invention relates to recombinant vectors whichcomprise the cDNA or amplified nucleic acid molecules of the presentinvention, to host cells which are genetically engineered with therecombinant vectors, to methods for the production of a recombinantpolypeptide using these vectors and host cells, and to recombinantpolypeptides produced using these methods.

Recombinant vectors may be produced according to this aspect of theinvention by inserting, using methods that are well-known in the art,one or more of the cDNA molecules or amplified nucleic acid moleculesprepared according to the present methods into a vector. The vector usedin this aspect of the invention may be, for example, a phage or aplasmid, and is preferably a plasmid. Preferred are vectors comprisingcis-acting control regions to the nucleic acid encoding the polypeptideof interest. Appropriate trans-acting factors may be supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression (and are therefore termed “expression vectors”),which may be inducible and/or cell type-specific. Particularly preferredamong such vectors are those inducible by environmental factors that areeasy to manipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids, and will preferablyinclude at least one selectable marker such as a tetracycline orampicillin resistance gene for culturing in a bacterial host cell. Priorto insertion into such an expression vector, the cDNA or amplifiednucleic acid molecules of the invention should be operatively linked toan appropriate promoter, such as the phage lambda P_(L) promoter, the E.coli lac, trp and tac promoters. Other suitable promoters will be knownto the skilled artisan.

Among vectors preferred for use in the present invention include pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; pcDNA3 available from Invitrogen; pGEX, pTrxfus,pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 available fromPharmacia; and pSPORT1, pSPORT2 and pSV.SPORT1, available from LifeTechnologies, Inc. Other suitable vectors will be readily apparent tothe skilled artisan.

The invention also provides methods of producing a recombinant host cellcomprising the cDNA molecules, amplified nucleic acid molecules orrecombinant vectors of the invention, as well as host cells produced bysuch methods. Representative host cells (prokaryotic or eukaryotic) thatmay be produced according to the invention include, but are not limitedto, bacterial cells, yeast cells, plant cells and animal cells.Preferred bacterial host cells include Escherichia coli cells (mostparticularly E. Coli strains dH10B and Stbl2, which are availablecommercially (Life Technologies, Inc.; Rockville, Md.)), Bacillussubtilis cells, Bacillus megaterium cells, Streptomyces spp. cells,Erwinia spp. cells, Klebsiella spp. cells and Salmonella typhimuriumcells. Preferred animal host cells include insect cells (mostparticularly Spodoptera frugiperda Sf9 and Sf21 cells and TrichoplusaHigh-Five cells) and mammalian cells (most particularly CHO, COS, VERO,BHK and human cells). Such host cells may be prepared by well-knowntransformation, electroporation or transfection techniques that will befamiliar to one of ordinary skill in the art.

In addition, the invention provides methods for producing a recombinantpolypeptide, and polypeptides produced by these methods. According tothis aspect of the invention, a recombinant polypeptide may be producedby culturing any of the above recombinant host cells under conditionsfavoring production of a polypeptide therefrom, and isolation of thepolypeptide. Methods for culturing recombinant host cells, and forproduction and isolation of polypeptides therefrom, are well-known toone of ordinary skill in the art.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLE 1

Cloning and Expression of RSV RNase H⁻ RT

General Methods

RSV H⁻ RT is a cloned form of retrovirus RT, in which both the α and theβ subunits are mutated by a single amino acid change to eliminate RNaseH activity. An RSV RT exhibiting substantially reduced RNase H activityis also produced when only the α subunit is mutated to substantiallyreduce RNase H activity (with the β subunit not being mutated in theRNase H domain). Mutations and plasmid constructions were conductedusing standard molecular biology methods (see, e.g., Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, N.Y.: Laboratory Press (1989)), modified as described below.

Plasmid Preparatiom

Plasmid preparations from 1 ml E. coli cultures were made by thealkaline Iysis procedure (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Laboratory Press(1989)). From 10 ml cultures, the preparation was additionally treatedwith phenol-chloroform, precipitated with ethanol, and resuspended in 50μl of Tris-EDTA (TE) buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0). Forbacmid preparations, care was taken to avoid shear of the DNA duringhandling (i.e., no vortexing; phenol-chloroform extractions were gentlyrolled rather than shaken; slow pipetting).

PCR

Polymerase chain reactions were carried out in a Perkin-Elmer 9600thermocycler. Reaction mixtures (50 μl each) contained 0.5 units of TaqDNA polymerase, 1 μM of each oligonucleotide, 50 μM each of dCTP, dGTP,dTTP, and dATP, and about 100 ng of target DNA in a reaction bufferconsisting of 50 mM KCl, 20 mM Tris-HCl (pH 8.3) and 5 MM MgCl₂. Unlessotherwise noted, the cycling conditions for each PCR were 5 minutes at94° C., followed by eight cycles of 15 seconds at 55° C./30 seconds at72° C./15 seconds at 94° C., and then 1 minute at 72° C.

Gel Electrophoresis and DNA Fragment Isolation and Cloning.

DNA was electrophoresed at about 10 V/cm in agarose gels inTris-acetate-EDTA buffer (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual 2nd ed., Cold Spring Harbor, N.Y.: Laboratory Press(1989)) containing about 0.3 μg/ml ethidium bromide. Fragments werevisualized with ultraviolet light and isolated from gel slices by theGlassMAX method (Simms, D., et al., Focus 13:99 (199 1)). DNA ligationreactions were performed using T4 DNA ligase under standard conditions(King, P. W., and Blakesley, R. W., Focus 8:1 (1986)), and E. coli dH10Bcells were transformed by a modification of the CaCl₂ method (Lorow, D.,and Jessee, J., Focus 12:19 (1990)).

Insect Cell Culture and Baculovirus Production

Samples (1 ml) of Sf21 insect cells at 5×10⁵ cells/ml were transfectedwith a mixture of 1 μg of bacmid DNA and 8 μl of Cellfectin in 0.2 mlSF900-II serum-free insect cell culture medium (Life Technologies, Inc.;Rockville, Md.; see Godwin, G., and Whitford, W., Focus 15:44 (1993)),according to published procedures (Anderson, D., et al., Focus 16:53(1995)). For growth and propagation, Sf9 and Sf21 insect cells werepassaged in SF900-lI medium at 27° C. in a shaking incubator at 100 rpm(for 600 ml cultures) or 130 rpm (for all other cultures). Care wastaken to avoid allowing the cultures to exceed 4×10⁶ cells/ml duringgrowth or to drop below 0.5×10⁶ cells/ml during dilution. To expandviral populations, Sf21 cells at about 1×10⁶ cells/ml were infected withenough viral stock (about 0.2% (v/v) virus/culture) to allow growth toabout 2×10⁶ cells/ml, but not more than 4×10⁶ cells/ml. After 72 hours,the culture was centrifuged (2,000 rpm for 10 min) and the supernatantwas decanted and stored in the dark at 4° C. For infection of cells forprotein production, Sf21 insect cells at about 1.5×10⁶ cells/ml wereinfected with enough virus to allow no growth or growth to less than2.5×10⁶ cells/ml. After 72 hours, the culture was harvested bycentrifugation at 1,000 rpm for 5 minutes and cells were resuspended in15 ml of PBS (0.2 g/liter KCl, 0.2 g/liter KH₂PO₄, 8 g/liter NaCl, 1.15g/liter Na₂HPO₄, 2.16 g/liter Na₂HPO₄.7H₂O) per 500 ml culture.

Cloning and Expression of Genes Encoding the RSV RT α and β Subunits

Both the α and β subunits of RSV RT are produced by proteolyticprocessing of larger polypeptide precursors (Gerard, G. F., in: Enzymesof Nucleic Acid Synthesis and Modification, Vol. I:DNA Enzymes, Jacob,S. T., ed., Boca Raton, Fla.: CRC Press, pp. 1-38 (1983)). To obviatethe requirement for proteolytic processing, the coding sequence for RSVRT was mutagenized and subcloned such that both the α and β subunitswere encoded by genes with standard start and stop translationalsignals. Both genes were mutagenized in the RNase H region, althoughconstruction of any combination of subunits (e.g., α RNase H⁻/β RNaseH⁺; α RNase H⁺/β RNase H⁺; α RNase H⁺/β RNase H⁻; α RNase H⁻/β RNase H⁻)may be accomplished in this same manner. It has been discovered that RSVRT α RNase H⁻/β RNase H⁺ is substantially reduced in RNase H activity(approximately 5% of wildtype). A sequence encoding an affinity tag wasadded to the carboxy end of the β subunit.

Mutagenesis and Subcloning of the Amino End, the Carboxy End and theMiddle of the RSV RT β Subunit

The RSV RT gene was mutagenized to add an ATG codon and an NdeI site tothe amino end of the sequence coding for the mature RT polypeptide. Thismutagenesis was accomplished by PCR using a pJD100 target (FIGS. 1, 7)(Wilkerson, V. W., et al., J. Virol. 55:314-321 (1985)) and thefollowing oligonucleotides:

AUG GAG AUC UCU CAT ATG ACT GTT GCG CTA CAT CTG GCT  (SEQ ID NO:1)

AAC GCG UAC UAG U GTT AAC AGC GCG CAA ATC ATG CAG  (SEQ ID NO:2)

PCR was performed, and PCR products purified, as described above and thePCR products were cloned into pAMP18 by UDG cloning (Buchman, G. W., etal., Focus 15:36 (1993)) to form plasmid pAMP18N (FIGS. 1, 8).

Following mutagenesis and cloning of the amino end, the carboxy end ofthe gene for the β subunit of RSV was mutagenized and subcloned fromPJD100 by PCR and UDG cloning into pAMP1 (FIG. 1), using the followingoligonucleotides:

CUA CUA CUA CUA GGT ACC CTC TCG AAA AGT TAA ACC  (SEQ ID NO:3)

CAU CAU CAU CAU CTC GAG TTA TGC AAA AAG AGG GCT CGC CTC ATC  (SEQ IDNO:4).

These oligonucleotides were designed to introduce a translational stopcodon in the β gene at the site in which the “p4” region was cleavedfrom the βp4 polypeptide, and to add an XhoI site after the end of thegene. The PCR products were purified by gel electrophoresis and clonedinto pAMP1 by UDG cloning, forming pAMP1C (FIGS. 1, 9). Note that thiscarboxy end is without a His tag, which was added later to form thefinal construct.

To add the middle region of the RSV RT β subunit, the 2.3 kb HpaI-KpnIfragment from pJD100 (FIG. 1) that encodes the middle of the β subunitof RSV RT was cloned into the HpaI-KpnI sites of pAMP18N, formingpAMP18NM (FIGS. 1, 10). To add the carboxy end of the RSV RT β subunit,the 113 bp KpnI-EcoRI fragment encoding the carboxy end of the β subunitgene was cloned from pAMP1C into the KpnI-EcoRI sites of pAM18NM,forming pAMP18B (FIGS. 2, 11).

Following construction of the RSV RT β subunit, which contains RNase Hactivity, this gene was mutagenized by site-directed mutagenesis toproduce a construct encoding a RSV RT β subunit that is substantiallyreduced in RNase H activity (i.e., “RNase H⁻”). A 3 Kb PstI fragmentfrom pJD100 (FIG. 2) containing the entire RT gene was cloned intoM13mp19, forming M13RT (FIGS. 2, 12). Single-stranded DNA containinguracil was isolated from E. coli strain CJ236 (Bio-Rad; Hercules,Calif.; and Cathy Joyce, Yale University, New Haven, Conn.) afterinfection with M13RT phage containing the PstI fragment. To mutate theRNase H region and to introduce an SstII site, the followingoligonucleotide was used:

GGA CCC ACT GTC TTT ACC GCG GCC TCC TCA AGC ACC  (SEQ ID NO:5)

This oligonucleotide induced the substitution of an alanine residue inplace of the aspartate residue at position 450 of the RT, formingM13RTH⁻ (FIGS. 2, 13). In an alternative approach to generate a RNase H⁻RSV RT, Glu484 may be mutated to glutamine and/or Asp505 may be mutatedto asparagine. To convert the β subunit back to RNase H⁺, anoligonucleotide primer having the wildtype sequence may be used.Alternatively, deletions or insertions can be made in the RNase H⁻ gionto substantially reduce RNase H activity. DNA sequencing was used toconfirm the Asp450→Ala450 mutation, and the 426 bp BglII-BstEII fragmentfrom M13RTH⁻ was cloned into the BglII-BstEII sites of pAMP18B,replacing the RNase H region and forming pAMP18BH− (FIGS. 2, 14).

Mutagenesis and Subcloning of the Gene Encoding the RSV RT α Subunit

To create a gene which codes for the α subunit of RSV RT, twooligonucleotides were used to mutagenize the amino end of the RNase H⁻mutant RSV RT gene from pDBH− (FIGS. 3, 15) and to introduce atranslational stop codon where avian retroviral protease p15 normallycleaves the precursor polyprotein to make the α subunit:

CAU CAU CAU CAU CCC GGG TTA ATA CGC TTG GAA GGT GGC  (SEQ ID NO:6)

 CUA CUA CUA CUA TCA TGA CTG TTG CGC TAC ATC TG  (SEQ ID NO:7)

PCR cycling conditions were 5 minutes at 94° C., followed by 8 cycles of15 seconds at 55° C./2 minutes at 72° C./15 seconds at 94° C., and then2 minutes at 72° C. The PCR products were cloned into pAMP1 by UDGcloning as described above, forming pAMP1A (FIGS. 3, 16).

Addition of a His₆ Tag to the Carboxy End of the RSV β Subunit

A His₆ tag was added to the carboxy end of the RSV RT β subunit bysite-directed mutagenesis, and the mutant sequence was subcloned frompJD100 by PCR and UDG cloning as described above, using the followingoligonucleotides:

CUA CUA CUA CUA GGT ACC CTC TCG AAA AGT TAA  (SEQ ID NO:8)

CAU CAU CAU CAU GAG GAA TTC AGT GAT GGT GAT GGT GAT GTG CAA AAAGAGG  (SEQ ID NO:9)

These oligonucleotides were designed to introduce a translational stopcodon in the gene, and to add a histidine tag to the end of the protein.The gene product was thus a polypeptide to which 6 histidines were addedto the carboxy end. The PCR products were purified by gelelectrophoresis and inserted into pAMP1 by UDG cloning, formingpAMPChis.

To remove the carboxy end of the RSV RT β gene and replace it with thecarboxy end containing a His₆ tag, the number of KpnI sites in thebaculovirus vector containing the RSV RT β gene had to be reduced.Plasmid pDBH−(FIG. 4) was cleaved with AflII, and the site was “blunted”with the Klenow fragment of E. coli DNA polymerase I, the polymerase wasinactivated by heat treatment, and the vector was further cleaved withPvuII (FIG. 4). The deleted vector (5.9 kb) was then purified by gelelectrophoresis, ligated to itself and used to transform E. coli straindH10B, forming pDBH-Kpn (FIGS. 4, 17). The KpnI-SstI fragment with thecarboxy end of β with the His₆ tag was cloned from pAMPChis into theKpnI-SstI sites of pDBH-Kpn, forming pDBH-KpnHis (FIGS. 4, 18).

Cloning of RSV α and β Subunits into a Baculovirus Expression Vector

During the course of this work, a construct was made in which the RSV RTβ gene was to be expressed in a baculovirus vector with a His₆ tag atthe amino end. However, a mutation introduced during the constructioncaused the reading frame of the tag to be different from that of the βgene. Since portions of this construct were used to construct the finalbaculovirus expression vector, its construction is described here.

To introduce a His₆ tag and an NdeI site into the baculovirus vector,the following oligonucleotides were annealed and cloned into pFastBacDual (Harris, R., and Polayes, D. A., Focus 19:6-8 (1997); FIG. 5):

ACTG GAA TTC ATG CCA ATC CAT CAC CAT CAC CAT CAC CCG T  (SEQ ID NO:10)

ACGT GTC GAC CAT ATG GAT GAC TAG GTG AAA CGG GTG ATG G  (SEQ ID NO:11)

Both oligonucleotides were formulated into TE buffer at a concentrationof 100 μM, and 5 μl of each oligonucleotide formulation were mixed with15 μl of water and 2 μl of 10×React2 buffer (500 mM NaCl, 500 mMTris-HCl, 100 mM MgCl₂, pH 8.0) in a single tube. This tube was heatedin a 65° C. water bath and cooled slowly over 60 minutes to 25° C. Theresulting product was a double-stranded DNA molecule with an EcoRI siteat the 5′ end (underlined), and a Sall site at the 3′ end (italicized):

5′-ACTG GAA TTC ATG CCA ATC CAT CAC CAT CAC CAT CAC CCG TTT CAC CTA GTCATC CAT ATG GTC GAC ACGT-3′  (SEQ ID NO:12)

This product was cleaved with EcoRI and SalI and the desired fragmentwas cloned into the EcoRI-SalI sites of vector pFastBac Dual by standardtechniques (Harris, R., and Polayes, D. A., Focus 19:6-8 (1997)),resulting in the formation of plasmid pFastBac Dual Nde (FIG. 5). TheNdeI-XhoI B fragment of pAMP18BH⁻ was cloned into the NdeI-SalI sites ofpFastBac Dual Nde to create pDBH⁻(FIGS. 5, 15).

To clone the RSV RT α gene into a baculovirus vector, the α gene wasexcised from pAMP1A with SmaI and BspHI and subcloned into the NcoI-SmaIsites of pFastBac Dual, creating pDA (FIGS. 5, 19). This placed the RSVRT α gene downstream from the baculovirus P10 promoter. The RSV αpeptide gene and P10 promoter were excised with RsrII and SmaI, andcloned into the RsrII-SmaI sites of pDBH−, forming pDABH− (FIGS. 4, 20).

To replace the RSV RT β gene in pDABH−with the carboxy His₆-tagged βgene in pDBH-KpnHis, pDBH-KpnHis was cleaved with NdeI, the siteblunt-ended with Klenow fragment, and the β gene with the carboxy His₆tag was then released with SstI (FIG. 6). pDABH⁻ was cleaved with EcoRI,the site blunt-ended with Klenow fragment, and then the β gene wasremoved by digesting with SstI. The vector fragment, which was ablunt-end SstI fragment of pFastBac Dual with the α a gene cloned infront of the p10 promoter, was ligated to the blunt-end SstI fragmentwith the carboxy His-tagged β gene from pDBH-KpnHis. E. coli dH10B cellswere transformed with this construct, and a transformant containingpDABH-His (FIGS. 6, 21) was selected.

The recombinant host cell comprising plasmid pDABH-His, E. colidH10B(pDABH-His), was deposited on Apr. 15, 1997, with the Collection,Agricultural Research Culture Collection (NRRL), 1815 North UniversityStreet, Peoria, Ill. 61604 USA, as Deposit No. NRRL B-21679.

Using the deposited plasmid, one of ordinary skill in the art may easilyproduce, using standard genetic engineering techniques (such as thosefor site-directed mutagenesis described above), plasmids encodingvarious forms of the α and/or β subunits of RSV RT (e.g., a RNase H⁺/βRNase H⁺; α RNase H⁻/β RNase H⁻; α RNase H⁺/β RNase H⁻; and α RNase H⁻/βRNase H⁺).

Transfection of Insect Cells for Virus and RSV RT Production

To prepare vectors for the transfection of insect cells, it was firstnecessary to insert the RSV RT gene constructs into a baculovirusgenome. One method of accomplishing this insertion is by using thesite-specific transposon Tn7 (Luckow, V. A, in: Recombinant DNATechnology and Applications, Prokop, A., et al., eds., New York:McGraw-Hill (1991)). In dH10Bac host cells, most of the baculovirusgenome is represented on a low copy plasmid (a “bacmid”) which alsocontains a Tn7 insertion site within a gene (Harris, R., and Polayes, D.A., Focus 19:6-8 (1997)). Transposition of Tn7 is facilitated by the Tn7transposase, which is produced from a second plasmid in dH10Bac hostcells, and pFastBac DUAL is constructed with the “right” and “left”regions of Tn7 flanking the promoter and cloning sites.

To insert the present constructs into a bacmid expression vector,pDABH-His was transformed into dH10Bac cells and transposition ofsequences encoding the RSV RT β gene (whose synthesis is directed by thebaculovirus polyhedrin promoter) and the RSV RT α gene (whose synthesisis directed by the baculovirus P10 promoter) was followed by screeningfor loss of β-galactosidase activity following transposition to the siteon the bacmid which codes for the β-galactosidase α peptide (Harris, R,and Polayes, D. A., Focus 19:6-8 (1997)). Bacmid DNA was then preparedfrom 10 ml transformant cultures by a slight modification of a standardminiprep procedure (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1989)). About 1 μg of the bacmid DNA was then used totransfect Sf21insect cells using the cationic lipid Cellfectin(Anderson, D., et al., Focus 16:53 (1995)).

For expansion of primary virus, the supernatant was removed from thetransfected cells about 72 hours after transfection and 1 ml was used toinfect 35 ml of Sf21 insect cells (about 1.2×10⁵ cells/ml). After 72hours, the culture was centrifuged (2,000 rpm; 10 min) and thesupernatant was decanted and used as a secondary viral stock. Thesecondary virus stock was expanded similarly by infecting 35 ml of Sf21cells with 0.1 ml of the secondary stock.

Virus stocks were then used to infect Sf21 cells for the expression ofRSV RT. In preliminary experiments, expression of RT activity frominfected cells was found to be maximal about 72 hours after infection.For test expressions, 70 ml of Sf21 cells were infected with 5 ml of thevirus stock, and the cells were harvested 72 hours after infection bycentrifugation at 1,000 rpm for 5 minutes, resuspension in PBS (2.5% ofthe culture volume) and recentrifugation at 1,000 rpm for 5 minutes.Supernatants were removed and the cells were stored. at −70° C. untiluse. For larger scale production, 600 ml of cells in 2.8 liter Fernbachflasks were infected with 2 ml of virus stock, and the cells wereharvested 72 hours after infection.

EXAMPLE 2

Isolation of RSV RNase H⁻ RT

To provide purified recombinant RSV H⁻ RT, cloned RSV H⁻ RT wasoverexpressed in cultured insect cells as described in Example 1 andpurified by affinity and ion-exchange chromatography. The RSV RTproduced comprises the α and β subunits. Isolation of RSV RT provides asubstantially pure RSV RT in which contaminating enzymes and otherproteins have been substantially removed, although such contaminantsneed not be completely removed.

Buffers

The pH of all buffers was determined at 23° C., and buffers were storedat 4° C. until use. Crack Buffer contained 50 mM Tris-HCl (pH 7.9), 0.5M KCl, 0.02% (v/v) Triton X-100 and 20% (v/v) glycerol. Just before use,the following protease inhibitors (Boehringer Mannheim; Indianapolis,Ind.) were added to Crack Buffer at the final concentrations indicated:leupeptin (2 μg/ml), Pefabloc (48 μg/ml), pepstatin A (2 μg/ml),benzmamidine (800 μg/ml) and PMSF (50 μg/ml). Buffer A contained 20 mMTris-HCl (pH 7.9), 0.25 M KCl, 0.02% (v/v) Triton X-100, and 10% (v/v)glycerol. Buffer B was Buffer A with 1 M imidazole added. Buffer Scontained 50 mM Tris-HCl (pH 8.2), 0.02% (v/v) Triton X-100, 10% (v/v)glycerol, 0.1 mM EDTA and 1 mM dithiothreitol (DTT). Buffer T was BufferS with 1 M KCl added. Buffer H contained 20 mM potassium phosphate (pH7.1), 0.02% (v/v) Triton X-100, 20% (v/v) glycerol, 0.1 mM EDTA and 1 mMDTT. Buffer J was Buffer H with 1 M KCl added. Storage Buffer contained200 mM potassium phosphate (pH 7.1), 0.05% (v/v) NP-40, 50% glycerol(v/v), 0.1 mM EDTA, 1 mM DTT, and 10% (w/v) trehalose.

Extract Preparation

Frozen insect cells (25 g) were thawed and a slurry prepared at 4° C.with 50 ml of Crack Buffer plus inhibitors. Cells were disrupted at 4°C. by sonication with a Fisher 550 Sonicator at 25% of maximum power.The disrupted crude extract was clarified by centrifugation at 27,000×gfor 30 minutes at 4° C.

RSV RT Isolation

Following clarification, the extract was fractionated and RSV RTpurified by column chromatography at 4° C. The clarified crude extractwas loaded unto a 30 ml Chelating Sepharose Fast Flow column (Pharmacia;Piscataway, N.J.) charged with NiSO₄ as per manufacturer's instructionsand equilibrated in 99.5% Buffer A+0.5% Buffer B. The column was washedwith one column volume of 99% Buffer A+1% Buffer B and then with 10column volumes of 98.5% Buffer A+1.5% Buffer B. RT was eluted with a10-column volume linear gradient of 98.5% Buffer A+1.5% Buffer B to 75%Buffer A+25% Buffer B.

During purification, reverse transcriptase activity was assayed withpoly(C)-oligo(dG), which is specific for reverse transcriptase (GerardG. F., et al., Biochemistry 13:1632-1641 (1974)). RT unit activity wasdefined and assayed as described (Houts, G. E., et al., J. Virol.29:517-522 (1979)). Using the poly(C)-oligo(dG) assay, the peakfractions of RT activity from the Chelating Sepharose Fast Flow column(10 to 17% Buffer B) were pooled, diluted with an equal volume of BufferS, and loaded on a 5 ml AF-Heparin-650 M column (TosoHaas,Montgomeryville, Pa.) equilibrated in Buffer S. After a wash with 12column volumes of 90% Buffer S+10% Buffer T, the column was eluted witha 15-column volume linear gradient of Buffer S to 30% Buffer S+70%Buffer T. The peak fractions of RT activity (43 to 50% Buffer T) werepooled, diluted with 2.5 volumes of Buffer H, and loaded unto a Mono SHR 5/5 column (Pharmacia, Piscataway, N.J.) equilibrated in Buffer H.After a wash with 20 column volumes of 85% Buffer H+15% Buffer J, thecolumn was eluted with a 20 column volume gradient of 85% Buffer H+15%Buffer J to 50% Buffer H+50% Buffer J. The RT peak fractions werepooled, dialyzed against Storage Buffer overnight, and stored at −20° C.

Following purification, RSV H⁻ RT was found to be >95% homogeneous asjudged by SDS-PAGE. The purified enzyme was also found to besubstantially lacking in RNase and DNase contamination, andsubstantially reduced in RNase H activity.

EXAMPLE 3

Preparation of Full-length cDNA Molecules

Enzymes

SuperScript II RT (SS II RT), a cloned form of Moloney murine leukemiavirus (M-MLV) RT lacking demonstrable RNase H activity (i.e., an “RNaseH⁻ RT”), was from Life Technologies, Inc. (Rockville, Md.). M-MLV RT, acloned murine RT with full RNase H activity (i.e., an “RNase H⁺ RT”),was also from Life Technologies, Inc. AMV RT, an RNase H⁺ uncloned formof avian myeloblastosis virus RT, was from Seikagaku America, Inc. RSVH⁻ RT was prepared as described in Examples 1 and 2. Recombinant Tth DNApolymerase, a cloned, thermophilic DNA polymerase from Thermusthermophilus with reverse transcriptase activity, was from Perkin Elmer.

Synthetic mRNA

A 7.5 kilobase (Kb) synthetic mRNA with a 120-nucleotide 3′ poly(A) tail(Life Technologies, Inc.; Rockville, Md.) was used as template to testthe efficiency of various enzymes alone or in combination.

cDNA Synthesis Reaction Mixtures

Reaction mixtures (20 μl each) contained the following components unlessspecified otherwise: 50 mM Tris-HCl (pH 8.4 at 24° C.), 75 mM KCl, 10 mMdithiothreitol, 1 mM each of [³²P]dCTP (300 cpm/pmole), dGTP, dTTP, anddATP, 25 μg/ml (p(dT)₂₅₋₃₀), 125 μg/ml of 7.5 Kb mRNA, and 35 units ofcloned rat RNase inhibitor. Reaction mixtures with RTs alone or incombination contained the following:

SS II RT alone: 0.5 mM dNTPs, 3 mM MgCl₂, and 200 units of SS II RT RSVH⁻ RT alone: 7.5 mM MgCl₂ and 7 units of RSV H⁻ RT; AMV RT alone: 50 mMKCl, 10 mM MgCl₂, 4 mM sodium pyrophosphate and 14 units of AMV RT SS IIRT plus RSV H⁻ RT: 5 mM MgCl₂, 200 units of SS II RT, and 7 units of RSVH⁻ RT; Tth DNA Polymerase 0.5 mM dNTPs, 1 mM MnCl₂ and 5 units of alone:Tth DNA Polymerase M-MLV H⁺ RT alone: 0.5 mM dNTPs, 3 mM MgCl₂, 50 μg/mlactinomycin D and 200 units of M-MLV H⁺ RT Tth DNA Polymerase plus sameas Tth DNA Polymerase alone, plus either SS II RT or RSV H⁻ either 200units of SS II RT or 7 units of RT: RSV H⁻ RT M-MLV H⁺ RT plus SS IIsame as M-MLV H⁺ RT alone, plus 200 RT: units of SS II RT; M-MLV H⁺ pluseither 5 mM MgCl₂, 50 μg/ml actinomycin D, 200 AMV RT or RSV H⁻ RT:units of M-MLV H⁺ RT, and either 14 units of AMV RT or 7 units of RSV H⁻RT AMV RT plus either SS II 5 mM MgCl₂, 50 μg/ml actinomycin D, 14 RT orRSV H⁻ RT: units of AMV RT, and either 200 units of SS II RT or 7 unitsof RSV H⁻ RT

When RTs were used in combination, one enzyme was added first followedimmediately by the addition of an aliquot of the second enzyme. In somecases in which a single enzyme was used, a second aliquot of the sameenzyme was added as a control to assess the effect of doubling theamount of the single enzyme.

All cDNA synthesis reactions were carried out at 45° C. for 50 minutes,and the resultant cDNA product was detectably labeled by theRT-catalyzed incorporation of a ³²P-labeled deoxyribonucleosidetriphosphate precursor. The total yield of cDNA was determined by acidprecipitation of a portion of the cDNA product and counting it in ascintillation counter. The ³²P-labeled cDNA product in the remainder ofthe reaction mixture was fractionated by alkaline agarose gelelectrophoresis (Carmichael, G. G., and McMaster, G. K., Meth. Enzymol.65:380-385 (1980)). The gel was dried and the size distribution of thecDNA product was established by autoradiography. Using theautoradiographic film as a template, the dried gel was cut and analyzedby scintillation counting to establish the fraction of full length (7.5Kb) product synthesized.

Tables 1 and 2 show the total amount of cDNA synthesized and full lengthcDNA synthesized, respectively, from the 7.5 Kb mRNA by enzymes alone orin combination. The following conclusions can be drawn:

1. When RTs were present alone, the highest yields of total and fulllength product were obtained with the RNase H⁻ forms of RT. With eitherRSV H⁻ RT or SS II RT, the total yield was almost double the highestyield obtained with an RNase H⁺ enzyme (1011 and 946 ng, respectively,versus 607 ng). The effect of removing RNase H from the reaction waseven more dramatic when fall length yields were examined. In this case,yields were at least tripled (234 and 208 ng for RSV H⁻ and SS II RTversus 79 and 26 for M-MLV H⁺ RT and AMV RT, respectively). Theseresults demonstrate the dramatic positive effect of eliminating RNase Hfrom RT.

2. When RTs were combined, several effects were observed. Mixing RTsfrom different sources, whether RNase H⁻ or RNase H⁺, increased totaland full length yields. This is consistent with the hypothesis thatpausing at sites unique to one enzyme can be reduced by a second RT witha different set of pause sites. However, by far the greatest yields oftotal and full length cDNA product were obtained when two differentRNase H⁻ RTs were combined (see shaded boxes in Tables 1 and 2). Theseresults indicate that the two RNase H⁻ enzymes cooperate to synthesizefull-length cDNA molecules: the first enzyme synthesizes truncated cDNAmolecules, which are then extended to full-length via the activity ofthe second enzyme. Thus, the compositions and methods of the presentinvention facilitate the synthesis of full-length cDNA molecules.

TABLE 1 Total Yield of cDNA Synthesized by Various Enzymes from 7.5-KbmRNA¹

¹Mass (ng) of total cDNA product ²not tested

TABLE 2 Yield of Full Length cDNA Synthesized by Various Enzymes from7.5 Kb mRNA¹

¹Mass (ng) of full-length (7.5 kb) cDNA product ²not tested

In the mixing experiments summarized in Tables 1 and 2, two reversetranscriptases were added simultaneously to a reaction under conditionsthat may have been suboptimal for a single given RT. This was the casewhen an avian and a murine RT were used together, since the MgCl₂concentration was set at 5 mM, between the optima of 3 mM and 7.5 mM formurine and avian RT, respectively. In addition, full advantage could notbe taken in these experiments of the thermal stability of avian RNase H⁻RT in reactions containing a less thermostable murine RT.

To use multiple enzymes and to address the fact that different enzymesmay have different optimal conditions, sequential additions or separateuse of the enzyme may be done in accordance with the methods of theinvention. For example, cDNA could be synthesized from differentaliquots of the same RNA in separate reaction tubes with different RTsunder reaction conditions optimal for each RT. Subsequently, the cDNAsfrom each reaction could be mixed before performing furthermanipulations. Alternatively, RTs could be used singly and sequentiallyin one tube to perform cDNA synthesis. That is, SuperScript II could beused first to copy an RNA population under optimal reaction conditions,and then conditions could be adjusted to optimal for RSV H⁻ RT in thesame tube, and further synthesis could be performed with the avian RT atelevated temperature.

EXAMPLE 4

Cloning and Expression of Avian Myeloblastosis Virus (AMV) RT and AMVRNase H⁻ (AMV H⁻) RT

General Methods

The AMV RT of the present invention is a cloned form of avian retrovirusRT. The AMV H⁻ RT is a variant of cloned AMV RT in which both the α andthe β subunits are mutated by a single amino acid change to eliminateRNase H activity, although AMV RT substantially reduced in RNase Hactivity is also produced by mutating the α subunit alone (the β subunitnot containing a mutation in the RNase H domain). Mutations and plasmidconstructions were conducted using standard molecular biology methods(see, e.g., Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor, N.Y.: Laboratory Press (1989)),modified as described below. Plasmid preparation, PCR, gelelectrophoresis, DNA fragment isolation and cloning, insect cell cultureand baculovirus production were all performed as described for RSV RTcloning and expression in Example 1.

Cloning and Expression of Genes Encoding the AMV RT α and β Subunits

To clone AMV RT, AMV viral RNA was prepared (Strauss, E. M., et al., J.Virol. Meth. 1:213 (1980)) from purified (Grandgenett, D. P., et al.,Appl. Microbiol. 26:452 (1973)) AMV obtained from Life Sciences (St.Petersburg, Fla.). AMV RT cDNA was prepared from AMV viral RNA with theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (LifeTechnologies, Inc.; Rockville, Md.) following the instructions in thekit manual. A primer specific for AMV RT was used that adds a NotI siteto the cDNA. Following preparation, AMV RT cDNA was cloned into pSPORT1that had been treated with SalI and NotI, resulting in a vector(pSPORT8) comprising the AMV RT gene (FIG. 22).

Both the α and β subunits of AMV RT are produced by proteolyticprocessing of larger polypeptide precursors (Gerard, G. F., in: Enzymesof Nucleic Acid Synthesis and Modification, Vol I: DNA Enzymes, Jacob,S. T., ed., Boca Raton, Fla.: CRC Press, pp. 1-38 (1983)). To obviatethe requirement for proteolytic processing, the coding sequence for AMVRT was mutagenized and subcloned such that both the α and β subunitswere encoded by genes with standard start and stop translationalsignals. To make RNase H⁻ constructs, both α and β genes weremutagenized in the RNase H region, although construction of anycombination of subunits (e.g., α RNase H⁻/β RNase H⁺; α RNase H⁺/β RNaseH⁺; α RNase H⁺/β RNase H⁻; α RNase H⁻/β RNase H⁻) may be accomplished inthis same manner. It has been discovered that AMV RT α RNase H⁻/β RNaseH⁺ is substantially reduced in RNase H activity (approximately 5% ofwildtype). A sequence encoding an affinity tag was added to the carboxyend of the β subunit.

Synthesis of cDNA from AMV RNA

The AMV RNA was copied into DNA using a 3′ primer which is complementaryto the 3′ end of the AMV RT gene, and which adds a NotI site to the 3′end of the gene (FIG. 22). The 5′ end of the resulting cDNA was madeinto a SalI end by the addition of a SalI adapter. This cDNA was thencloned into a SalI-NotI cleaved vector (PSPORT1). First strand cDNA wassynthesized using Superscript II RT (Gerard, G. F., et al., FOCUS 11:66(1989)) and a gene specific primer (Oligonucleotide #1) instead of theNotI primer-adapter:

Oligonucleotide #1 (SEQ ID NO:13)

5′ GACTAGTTCTAGATCGCGAGCGGCCGCCCATTAACTCTCGTTGG CAGC 3′

The second strand synthesis was achieved using DNA polymerase I incombination with E. coli RNase H and DNA ligase at 16° C. andsubsequently polishing the termini with T4 DNA polymerase. The cDNA wasdeproteinized and precipitated with ethanol, and SalI adaptersconsisting of Oligonucleotides #2 and #3 were ligated to the cDNA: psOligonucleotide #2 (SEQ ID NO:14)

5′ TCGACCCACGCGTCCG 3′

Oligonucleotide #3 (SEQ ID NO:15)

5′ CGGACGCGTGGG 3′

The addition of the adapters was followed by digestion with NotI. Sizefractionation of the cDNA was done on 1 ml prepacked columns providedwith the cDNA cloning kit. The amount of cDNA in each fraction wascalculated from the specific activity of incorporated ³²P label, and thesize of the cDNA was determined by autoradiography of an agarose gel.Those fractions that were greater than 3 Kb were selected for cloning.

Cloning AMV cDNA into a Vector

The cDNA was ligated into SalI-NotI cleaved pSPORT1 vector and then theligated cDNA was used to transform E. coli MAX EFFICIENCY dH10B™competent cells (Life Technologies, Inc., Rockville, Md.). Aftertransformation, aliquots of cells were plated on LB plates containingampicillin. Twelve colonies were picked and 1 ml cultures were grown formini-preps. Gels were run to check for certain fragments after digestingwith restriction enzymes SalI, MluI, PstI, ApaI, DraIII, SphI and BglII.One plasmid, pSPORT8, was selected since the insert was large enough tocode for the AMV RT gene (3 Kb) and a PstI site was present whichindicated that the 5′ terminus of the AM RT gene was present (FIG. 22).

Mutagenesis and Subcloning of the Amino End, the Carboxy End and theMiddle of the AMV RT β Subunit

The AMV RT gene was mutagenized to add an ATG codon and an EcoRI site tothe amino end of the sequence coding for the mature RT polypeptide byPCR with pSPORT8 as the target and the following oligonucleotides:

Oligonucleotide #4 (SEQ ID NO:16)

5′ AUG GAG AUC UCU GAA TTC ATG ACT GTT GCG CTA CAT CTG GCT 3′

Oligonucleotide #5 (SEQ ID NO:2)

5′ AAC GCG UAC UAG U GTT AAC AGC GCG CAA ATC ATG CAG 3′

PCR was performed, and PCR products purified, as described above. ThePCR reaction was treated with DpnI to destroy the target and the PCRproduct was cloned into pAMP18 by UDG cloning (Buchman, G. W., et al.,Focus 15:36 (1993)), forming plasmid pAMVN (FIGS. 23, 26).

Following mutagenesis and cloning of the amino end, a His₆ affinity tag,a XhoI site and a translational stop codon were added to the carboxy endof the gene for the β subunit of AMV RT in pSPORT8 by PCR using thefollowing oligonucleotides:

Oligonucleotide #6 (SEQ ID NO:3)

5′ CUA CUA CUA CUA GGT ACC CTC TCG AAA AGT TAA ACC 3′

Oligonucleotide #7 (SEQ ID NO:9)

5′ CAU CAU CAU CAU GAG GAA TTC AGT GAT GGT GAT GGT GAT GTG CAA A AAG AGG3′

PCR was performed, and PCR products purified, as described above. ThePCR reaction was treated with DpnI to destroy the target and the PCRproduct was cloned into pAMP18 by UDG cloning (Buchman, G. W., et al.,Focus 15:36 (1993), forming plasmid pAMVC (FIGS. 23, 27).

To add the middle region of the AMV RT β subunit, the 2.3 Kb HpaI-KpnIfragment from pSPORT8 that encodes the middle of the β subunit of AMV RTwas cloned into the HpaI-KpnI sites of pAMVN, forming pAMVNM (FIGS. 23,28). To add the carboxy end of the AMV RT β subunit, since pAMVC has twoKpnI sites (FIG. 27), it was partially cleaved with KpnI, thencompletely cleaved with ScaI, and the 3 Kb fragment with the carboxy endof AMV PT was isolated and ligated to the 3.5 Kb ScaI-KpnI fragment ofpAMVNMH− (FIG. 29), forming pAMVBH− (FIGS. 23, 30).

Mutagenesis of the Beta Subunit to RNase H⁻

The RSV RT and AMV RT genes are related (GenBank sequences J02342,J02021 and J02343 for RSV-C; L10922, L10923, L10924 for AMV). Thesegenes code for an identical sequence of amino acids over a shortdistance of the RNase H region. As described above in Example 1, duringthe cloning and mutagenesis of the RSV RT genes an RNase H⁻ derivativeof the RSV RT β gene was made by site- directed mutagenesis. Theoligonucleotide that was used (oligonucleotide #8) changed amino acidAsp450 to an Ala450 and introduced an SstII site (underlined).

Oligonucleotide #8 (SEQ ID NO:24)

The plasmid with the RNase H− mutation in the RSV RT β gene is pAMP18BH−(FIGS. 2, 14). The 129 bp BsrGI-BstEII fragment from pAMP18BH− wascloned into the BsrGI-BstEII sites of pAMVNM, replacing the RNase H⁺region in this plasmid and forming pAMVNMH⁻ (FIGS. 23, 29). The effectof this replacement was to change Asp450 to Ala450 in the AMV β genewithout changing any other amino acids. To convert the β subunit back toRNase H+, an oligonucleotide primer having the wildtype sequence may beused.

Mutagenesis and Subcloning of the Gene Encoding the AMV RT α Subunit

To create a gene which codes for the α subunit of AMV RT,oligonucleotides #9 and #10 were used to mutagenize the amino end of theAMV RT gene from pAMVNM to introduce a translational stop codon whereavian retroviral protease p15 normally cleaves the precursor polyproteinto make the α subunit:

Oligonucleotide #9 (SEQ ID NO:6)

5′ CAU CAU CAU CAU CCC GGG TTA ATA CGC TTG GAA GGT GGC 3′

Oligonucleotide #10 (SEQ ID NO:7)

5′ CUA CUA CUA CUA TCA TGA CTG TTG CGC TAC ATC TG 3′

PCR cycling conditions were 5 minutes at 94° C., followed by 8 cycles of15 seconds at 55° C./2 minutes at 72° C./15 seconds at 94° C., and then2 minutes at 72° C. The PCR reaction was treated with DpnI to destroythe target and the PCR product was cloned into pAMP18 by UDG cloning(Buchman, G. W., et al., Focus 15:36 (1993)), forming plasmid pAMVA(FIGS. 24, 31). To make the RNaseH⁻ allele of the α subunit of AMV RT,the same procedure was followed using pAMVBH− as a target, formingplasmid pAMVAH− (FIGS. 24, 32).

Cloning the AMV RT α and β Genes into pFastBac Dual

The α gene was excised from pAMVA with SmaI and BspHI, and subclonedinto the NcoI-PvuII sites of pFastBac Dual (pD; FIG. 33), creatingplasmid pDAMVA (FIGS. 25, 34). An RNase H⁻ AMV RT α gene was similarlycloned from pAMVAH−, forming plasmid pDAMVAH− (FIGS. 25, 35). In bothplasmids, the AMV RT α gene was downstream from the baculovirus P10promoter. The RNase H− AMV RT β gene was excised from pAMVBH− with EcoRIand cloned into the EcoRI site of pDAMVA. Clones were selected in whichthe EcoRI insert was oriented such that the AMV RT β gene was downstreamfrom the polyhedrin promoter, forming plasmid pDAMVABH− (FIGS. 25, 36).In this construct, the AMV RT α gene was RNase H⁺, but the β gene wasRNase H⁻. The AMV RNase H⁻ RT β gene and polyhedrin promoter wereexcised from pDAMVABH− with RsrII and NotI and cloned into theRsrII-NotI sites of pSPORT1, forming plasmid pJAMVBH− (FIGS. 25, 37).The AMV RNase H⁻ RT β gene and polyhedrin promoter were excised frompJAMVBH− with RsrII and NotI and cloned into the RsrII-NotI sites ofpDAMVAH−, forming plasmid pDAMVAH−BH− (FIGS. 25, 38).

The recombinant host cell comprising plasmid pDAMVABH−, E. colidH10B(pDAMVABH−), was deposited on Jun. 17, 1997, with the Collection,Agricultural Research Culture Collection (NRRL), 1815 North UniversityStreet, Peoria, Ill. 61604 USA, as Deposit No. NRRL B-21790.

Using the deposited plasmid, one of ordinary skill in the art may easilyproduce, using standard genetic engineering techniques (such as thosefor site-directed mutagenesis described above), plasmids encodingvarious forms of the α and/or β subunits of AMV RT (e.g., α RNase H⁺/βRNase H⁺; α RNase H⁻/β RNase H⁻; α RNase H⁺/β RNase H⁻; and α RNase H⁻/βRNase H⁺).

Transfection of Insect Cells for Virus and AMV RT Production

To prepare vectors for the transfection of insect cells, it was firstnecessary to insert the AMV RT gene constructs into a baculovirusgenome. This insertion was accomplished using the site-specifictransposon Tn7, as described for the insertion of the RSV RT geneconstructs into bacmids in Example 1. Plasmid pDAMVABH− was transformedinto dH10Bac cells and transposition of sequences encoding the AMV RT βgene (whose synthesis is directed by the baculovirus polyhedrinpromoter) and the AMV RT α gene (whose synthesis is directed by thebaculovirus P10 promoter) was followed by screening for loss ofβ-galactosidase activity following transposition to the site on thebacmid which codes for the β-galactosidase α peptide (Harris, R., andPolayes, D. A, FOCUS 19:6-8 (1997)). Bacmid DNA was then prepared fromtransformants (10 ml cultures) by a slight modification of a standardminiprep procedure, as described in Example 1. About 1 μg of the bacmidDNA was used to transfect Sf21 insect cells using the cationic lipidCellfectin (Anderson, D., et al., FOCUS 16:53 (1995)).

For expansion of primary virus, the supernatant was removed from thetransfected cells about 72 hours after transfection and 1 ml was used toinfect 35 ml of Sf21 insect cells (about 1.2×10⁵ cells/ml). After 72hours, the culture was centrifuged (2,000 rpm; 10 min) and thesupernatant was decanted and used as a secondary viral stock. Thesecondary virus stock was expanded similarly by infecting 35 ml of Sf21cells with 0.1 ml of the secondary stock.

Virus stocks were then used to infect Sf21 cells for the expression ofAMV RT. In preliminary experiments, expression of RT activity byinfected cells was found to be maximal about 72 hours after infection.For test expressions, 70 ml of Sf21 cells were infected with 5 ml of theviral stock, and the cells were harvested 72 hours after infection bycentrifugation at 1,000 rpm for 5 minutes, resuspension in PBS (2.5% ofthe culture volume) and recentrifugation at 1,000 rpm for 5 min.Supernatants were removed and the cells were stored at −70° C. untilused. For larger scale production, 600 ml of cells in 2.8 liter Fernbachflasks were infected with 2 ml of viral stock, and the cells wereharvested 72 hours after infection. AMV RT was then isolated asdescribed for RSV RT in Example 2.

EXAMPLE 5

Reverse Transcription with Retroviral RTs at Temperatures Above 55° C.

Retroviral reverse transcriptases have historically been used tocatalyze reverse transcription of mRNA at temperatures in the range of37° C. to 42° C. (see technical literature of commercial suppliers ofRTs such as LTI, Pharmacia, Perkin Elmer, Boehringer Mannheim andAmersham). There is a prevailing belief that at these temperatures mRNAsecondary structure interferes with reverse transcription (Gerard, G.F., et al., FOCUS 11:60 (1989); Myers, T. W., and Gelfand, D. H.,Biochem. 30:7661 (1991)) and the specificity of primer binding isreduced during gene-specific reverse transcription processes, such asRT-PCR, causing high background signal (Myers, T. W., and Gelfand, D.H., Biochem. 30:7661 (1991); Freeman, W. N., et al., BioTechniques20:782 (1996)). It is therefore desirable to carry out RNA reversetranscription at more elevated temperatures, i e., above 55° C., to helpalleviate these problems.

As noted above, retroviral RTs are generally not used at temperaturesabove 37° C. to 42° C. to copy RNA because of the limited thermalstability of these mesophilic enzymes. In recent years, however, it hasbeen reported that AMV RT can be used to perform RT-PCR of smallamplicons (<500 bases) at 50° C., and to a limited extent at 55° C.(Freeman, W. M., et al., BioTechniques 20:782 (1996); Mallers, F., etal., BioTechniques 18:678 (1995); Wang, R. F., et al., BioTechniques12:702 (1992)). Forms of M-MLV RT lacking RNase H activity, because ofremoval of the RNase H domain (Gerard, G. F., et al., FOCUS 11:66 (1989)or because of point mutations in the RT gene (Gerard, G. F., et al.,FOCUS 14:91 (1992)), can also be used at 50° C., but not at 55° C., tocatalyze cDNA synthesis.

Therefore, the thermal stability of RNase H⁻ RSV RT and its utility inhigher temperature (i. e., above 50° C.) reverse transcription reactionsfor synthesis of large cDNAs was examined.

Methods

Enzymes and RNAs

Superscript RT (SS RT), SuperScript II RT (SS II RT), and Moloney murineleukemia virus (M-MLV) RT were from LTI. AMV RT was from SeikagakuAmerica, Inc., or was prepared as described above in Example 4. SS RT isan RNase H⁻ form of M-MLV RT in which RNase H activity has beeneliminated by removing the RNase H domain of the RT polypeptide,resulting in an enzyme with a molecular weight of 57 KDa rather than 78KDa (Gerard, G. F., et al., FOCUS 11:66 (1989); Kotewicz, M. L., et al.Nuc. Acids Res. 16:265 (1988)). SS II RT is an RNase H⁻ form of M-MLV RTin which RNase H activity has been eliminated by the introduction ofthree point mutations in the RNase H domain of M-MLV RT (Gerard, G. F.,et al., FOCUS 14:91 (1992)). Rous Associated Virus (RAV) RT was fromAmersham. RSV RNase H⁻ and RSV RNase H⁺ RT were cloned, expressed andpurified as described above in Examples 1 and 2. The RNAs used astemplates were synthetic RNAs of 1.4, 2.4, 4.4 and 7.5 Kb, each with a120 nucleotide poly(A) tail at the 3′ end, obtained from LTI. SyntheticCAT mRNA was from LTI.

Model System for Determining Functional Thermal Stability of ReverseTranscriptases

A mixture of 1.4-, 2.4-, 4.4-, and 7.5-Kb mRNAs was used to test theability of various RTs to synthesize full-length cDNA copies at varioustemperatures. The cDNA products synthesized were labeled radioactivelyby the RT-catalyzed incorporation of a ³²P-labeled deoxyribonucleotidetriphosphate precursor. The ³²P-labeled cDNA products were fractionatedby alkaline agarose gel electrophoresis (Carmichael, G. G., andMcMaster, G. K., Meth. Enzymol. 65:380(1980)). The gel was dried and thesize distribution of the cDNA products was established byautoradiography. Using the autoradiographic film as a template, the fulllength cDNA bands at 1.4, 2.4, 4.4 and 7.5 Kb were cut from the driedgel and counted in scintillant to establish the amount of each fulllength product synthesized.

cDNA Synthesis Reaction Conditions

All cDNA synthesis reactions were carried out at the indicatedtemperatures for 30 or 50 minutes. All reaction mixtures were 20 μl andcontained the following components unless specified otherwise: 50 mMTris-HCl (pH 8.4 at 24° C.), 75 mM KCl, 10 mM dithiothreitol, 1 mM eachof [³²P]dCTP (300 cpm/pmole), dGTP, dTTP, and dATP, 25 μg/ml p(dT)₂₅₋₃₀,12.5 μg/ml each of 1.4-, 2.4-, 4.4-, and 7.5-Kb mRNA, and 35 units ofcloned rat RNase inhibitor. In addition, reaction mixtures contained thefollowing:

SS II RT: 0.5 mM dNTPs (instead of 1 mM), 3 mM MgCl₂ and 200 units of SSII RT RSV H⁻ RT: 7.5 mM MgCl₂ and 21 units of RSV H⁻ RT; AMV RT: 50 mMKCl, 10 mM MgCl₂, 4 mM sodium pyrophosphate and 29 units of AMV RT RSVH⁺ RT: 50 mM KCl, 10 mM MgCl₂, 4 mM sodium pyrophosphate and 24 units ofRSV H⁺ RT RAV RT: 50 mM KCl, 10 mM MgCl₂, 4 mM sodium pyrophosphate and24 units of RAV RT

Reaction mixtures containing all components except enzyme werepreincubated at the desired temperature for three minutes, and then RTwas added to initiate cDNA synthesis.

Half Life Determinations

The half lives of RTs were determined by incubating individual tubes ofRT at a desired temperature for appropriate lengths of time and stoppingthe incubation by placing the tube on ice. RSV RTs, RAV RT and AMV RTwere incubated in 20 μl aliquots containing 50 mM Tris-HCl (pH 8.4), 75mM KCl, 7.5 mM MgCl₂, 10 mM dithiothreitol, 50 μg/ml CAT mRNA, 25 μg/mlp(dT)₁₂₋₁₈, and 350-700 units/ml RT. Murine RTs (SS RT, SS II RT, M-MLVRT) were incubated in mixtures containing the same components exceptMgCl₂ was at 3 mM and enzyme was at 2,500 units/ml. An aliquot from eachtube (5 μl for avian RTs and 1 μl for murine RTs) was assayed for RTactivity in a unit assay reaction mixture to determine residual RTactivity.

Unit Assays

Unit assay reaction mixtures (50 μl) contained 50 mM Tris-HCl (pH 8.4),40 mM KCl, 6 mM MgCl₂, 10 mM dithiothreitol, 500 μM [³H]dTTP (30cpm/pmole), 0.5 mM poly(A), and 0.5 mM (dT)₁₂₋₁₈. Reaction mixtures wereincubated at 37° C. for 10 minutes and labeled products were acidprecipitated on GF/C glass filters that were counted in a scintillationcounter.

Results and Discussion

With a few exceptions, the half lives in the presence of a templateprimer at 45° C., 50° C., 55° C. and 60° C. were determined for RSV H⁺RT, RAV RT, AMV RT, RSV H⁻ RT, M-MLV H⁺ RT, SS RT, and SS II RT. Theresults are shown in FIG. 39 and Table 3.

TABLE 3 HALF LIVES OF REVERSE TRANSCRIPTASES¹ HALF LIFE (MINUTES) AT:ENZYME 45° C. 50° C. 55° C. 60° C. RSV H⁻ RT 440 138 5   0.75 RSV H⁺ RTND² 30 ND² ND³ RAV RT 159 37 3.8 ND³ AMV RT  96 16 1.3 ND³ SuperScriptII RT 105 7 ND³ ND³ SuperScript RT 120 3 ND³ ND³ M-MLV RT  65 2.5 ND³ND³ ¹Half lives were determined from the data shown in FIG. 39. ²ND: notdetermined. ³ND: not determined, because half life in this reactionmixture was too short to be determined accurately.

The results shown in FIG. 39 and Table 3 clearly demonstrate that RNaseH⁺ RTs (RSV, RAV and AMV) are much more thermostable than M-MLV RT, andhave reasonable half lives at 50° C. Furthermore, mutating these RTs toproduce their corresponding RNase H⁻ forms further increases their halflives. Most dramatically, RNase H⁻ RSV RT had a much longer half lifethan any other retroviral RT at 45° C., 50° C. and 55° C. Thus,introduction of a single amino acid change into the RNase H domain ofeach subunit of RSV RT increases its half life at 50° C. by nearlyfive-fold.

The impact of this increased thermal stability on cDNA synthesis attemperatures above 50° C. was found to be dramatic. FIG. 40 shows theresults of a comparison of the performance of these RTs at 45° C., 50°C., 55° C. and 60° C. in copying mRNA of 1.4 to 7.5 Kb in length. Withthe exception of RSV H⁻ RT, none of the RTs were found to producesubstantial product longer than 2.4 Kb in length at 55° C. or 60° C. RSVH⁻ RT, in contrast, continued to make full-length 7.5-Kb cDNA at 55° C.,and 4.4-Kb cDNA at 60° C. FIGS. 41 and 42 show a more detailedcomparison of the two RTs that performed best in FIG. 40 (i.e., SS II RTand RSV H⁻ RT). At temperatures above 55° C., RSV H⁻ RT was found tocontinue to synthesize cDNAs of all lengths, while SS II RT producedonly low levels of cDNA greater than 1 Kb in length.

Taken together, these results demonstrate that RNase H⁻ RSV RT is muchmore thermoactive than any other retroviral RT available commercially,and can be used to synthesize longer cDNAs (up to 4 Kb long) at 60° C.This enhanced thermoactivity of RNase H⁻ RSV RT is due to an increasedthermal stability, relative to RNase H⁺ RT, at 50° C. to 60° C., whichmakes RSV H⁻ RT an ideal enzyme for use in the reverse transcription ofmRNA at 50° C. to 60° C.

EXAMPLE 6

Reverse Transcription with Avian RTs at Elevated Temperatures

In Example 5 (Table 3 and FIG. 39), the half lives of various RTs werepresented. In particular, half lives were reported for cloned RSV RT inwhich the RNase H domain of each subunit was mutated to eliminate RNaseH activity (Example 1; Asp450→Ala in both the α subunit and the βsubunit).

To further examine the effects of these mutations on RT half life,constructs were produced as described above, in which only one of thetwo subunits were mutated at one time, such that various combinations ofmutants were formed (e.g., α RNase H⁻/β RNase H⁺ and α RNase H⁺/β RNaseH⁻). The half lives of these RSV RTs, as well as cloned AMV α RNase H⁻/βRNase H⁺ RT, were determined as described above in the Methods sectionof Example 5.

As shown in Table 4, when the α subunit in RSV or AMV RT was mutated,leaving the β subunit wild type intact, the resulting RT demonstratedgreater thermal stability than that observed for other avian RTs. Thesemutant RTs were also examined for their functional thermal stabilityusing the model system described in the Methods section of Example 5.For each enzyme, the increased thermal stability was found to correlatewith improved functional performance—for example, the α RNase H⁻/β RNaseH⁺ avian RTs, which demonstrated the highest thermal stability at 55° C.(Table 4), also demonstrated the highest functional activity at variouselevated temperatures (Table 5).

TABLE 4 Half Lives of RSV and AMV Reverse Transcriptases Enzyme HalfLife (Minutes) at 55° C. RSV αH⁻βH⁻ RT 5 RSV αH⁻βH⁺ RT 7 RSV αH⁺βH⁻ RT 2RSV αH⁺βH⁺ RT 1.9 AMV αH⁻βH⁺ RT 6 Native AMV RT 1.3 Native RAV RT 3.8

TABLE 5 Functional Activities of RSV RTs at Elevated Temperatures.Amount of Full-Length Temperature, Product Produced (pMoles) Enzyme ° C.1.4 Kb 2.4 Kb 4.4 Kb 7.4 Kb RSV αH⁻/βH⁻ RT 45.0 132.9 80.5 56.7 28.155.0 115.4 70.7 40.8 12.5 57.5 81.9 43.2 17.2 3.1 60.0 7.0 1.8 0 0 62.50 0 0 0 RSV αH⁻/βH⁺ RT 45.0 145.2 85.3 57.9 31.8 55.0 161.3 83.0 53.321.5 57.5 140.1 77.7 41.1 11.6 60.0 67.6 30.0 7.5 0.1 62.5 4.1 0.8 0 0

EXAMPLE 7

Alternative Methods of Generating Avian Reverse Transcriptases andCharacterization of their Properties

As noted above in the Related Art section, three prototypical forms ofretroviral RT have been studied thoroughly—M-MLV RT, HIV RT, and ASLV RT(which includes RSV and AMV RT). While each of these retroviral RTsexist as heterodimers of an α and a β subunit, there have been noreports heretofore of the simultaneous expression of cloned ASLV RT αand β genes resulting in the formation of heterodimeric αβ RT.

Examples 1-4 above described the cloning, expression and purification ofαβ forms of RSV and AMV RT that copy mRNA efficiently. Formation of αβRT was achieved in baculovirus-infected insect cells by co-expression ofgenes for α and β from a dual promoter vector. The studies presented inthis Example were designed to generate RSV αβ RT by a variety of othermethods that have been used to successfully clone and express HIVp66/p51 RT. In addition, as described below, the individual subunits ofRSV RT, including βp4, β and α, have now been cloned, expressed, andpurified, and the abilities of these subunits to copy mRNA has now beencharacterized.

Materials and Methods

General Methods

Mutations and plasmid constructions were conducted using standardmolecular biology methods (see e.g., Sambrook, J. et al., MolecularConing: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press (1989)), modified as described below.Plasmid preparation, PCR, gel electrophoresis, DNA fragment isolationand cloning, insect cell culture and baculovirus production were allperformed as described for RSV RT cloning and expression in Example 1.

Cloning and Expression of RSV RT in E. coli

A number of approaches were tried to generate RSV αβ RT from RSV RT βp4in E.coli.

PCR of NdeI-XbaI Fragment

The amino end of the RSV RT βp4 gene was mutagenized to introduce anNdeI site by PCR (1 cycle 94° C. for 5 min.; 15 cycles 94° C., 10 sec,55° C., 15 sec, 72° C., 15 sec.; 1 cycle 72° C., 5 min) of pJD100 (FIG.7) with the following oligonucleotides:

Oligonucleotide #11 (SEQ ID NO:17)

5′- ATT ATT CAT ATG ACT GTT GCG CTA CAT CTG GC-3′

Oligonucleotide #12 (SEQ ID NO:18)

 5′- TAC GAT CTC TCT CCA GGC CAT TTT C-3′

The NdeI site in oligonucleotide #11 above appears in bold anditalicized print, while the bases that are underlined inoligonucleotides #11 and #12 were derived from authentic RT genesequences. The PCR product with these two oligos contained an NdeI siteat the beginning of the gene, and retained the XbaI site which ispresent within the RT gene. The PCR product was cloned into pUC18between the NdeI and XbaI sites, forming pUC18#3.

Site-directed Mutagenesis to Introduce a SmaI Site to Clone p15

A 3 Kb PstI fragment from pJD100 containing the entire RSV RT gene wascloned into M13mp19. Clone RF-SmaI was then produced by introducing aSmaI site at the carboxy end of the RSV RT βp4 gene by site-directedmutagenesis (see Example 1), using the following mutagenicoligonucleotide:

Oligonucleotide #13 (SEQ ID NO:19)

5′- ACT CGA GCA GCC CGG GAA CCT TTG -3′

Reconstruction of the RT Gene.

The 2.8 kb SmaI-PstI fragment from RF-SmaI was cloned into pUC18 at theSmaI/PstI sites. The clone was designated as pUC18-PstI-SmaI. TheNdeI-XbaI fragment from pUC18 #3 was introduced into pUC18-PstI-SmaI toregenerate the entire RSV RT βp4 gene with an NdeI site at theinitiation codon. The clone was designated as pUC18-RT.

Cloning Entire RT Gene in pRE2

The NdeI-XbaI fragment from pUC18 #3 was cloned into expression vectorpRE2 to generate pRE2-Nde-Xba pRE2 contains an inducible lambda pLpromoter. The rest of the RSV RT βp4 gene was subcloned as an NdeI-SstIfragment from pUC18-PstI-SmaI into pRE2-Nde-Xba digested with XbaI andSstI, generating pRE2-RT.

Cloning of p15 into pRE2-RT

RSV RT is processed from RSV RT βp4 to the αβ heterodimer by the RSV p15protease. In order to make authentic αβ, the p15 protease gene wascloned and expressed with the RSV RT βp4 gene as follows. The RSV p15protease gene was generated by PCR using pJD100 as target and theprotocol described above. The oligonucleotides used for PCR were asfollows:

Oligonucleotide #14 (SEQ ID NO:20)

5′-AT TAC CCG GGA GG A TAT CAT ATG TTA GCG ATG ACA ATG GAA CAT AAA G -3′

Oligonucleotide #15 (SEQ ID NO:21)

5′- A TAT GTC GAC TCA CAG TGG CCC TCC CTA TAA ATT TG-3′

In oligonucleotides #14 and #15 above, the restriction sites (SmaI, NdeIand salI) are indicated in bold letters while the region of basesunderlined is the ribosome binding site. PCR using theseoligonucleotides generated a ˜450 bp fragment, which was digested withSmaI and SalI and cloned into pUC19. The clone was designated aspUC19-p15. The p15 gene was introduced into pRE2-RT by subcloning the450 bp SmaI-SalI fragment at the SmaI/SalI sites. The final plasmid wasdesignated pRE2-RT.p15.

Expression of RT from pRE2-RT and pRE2-RT.p15

E. coli CJ374 containing either pRE2-RT or pRE2-RT.p15 was grown at 30°C. in EG broth in the presence of ampicillin (100 μg/ml) andchloramphenicol (30 μg/ml) to an A590 of 0.5. Half of the culture wasinduced at 42° C. for 45 min., and then outgrown at 30° C. for 2 hr. Theother half was grown at 30° C. as an uninduced control. None of thecultures produced any visible induced protein upon examination bySDS-PAGE. None of the cell extracts displayed any RT activity.

Recloning of RT.p15 in pRE1

The lack of RT expression in the above constructs suggested that it waspossible that (i) a mutation had been introduced into the RT gene duringPCR to render it inactive, or (ii) during cloning a mutation in thelambda pL promoter arose, since RT is thought to be toxic to E. coli.Thus, the entire RT.p15 gene was recloned into pRE1, which was the sameas pRE2 except the multiple cloning site was in an opposite orientation,as a NdeI-SalI fragment and the construct was introduced into E. coliCJ374. In addition, a 2269 bp HpaI-KpnI fragment of the resulting clonewas replaced by the same HpaI-KpnI fragment from pJD100. Thisreplacement left only about a 200 bp amino terminal region derived fromPCR. Moreover, this region (NdeI site to HpaI site) was sequenced toconfirm that there was no mutation due to PCR. The plasmids weredesignated as pRE1-RT.15. This plasmid was also introduced into BL21, aprotease-deficient E. coli strain. The p15 gene was also deleted frompRE1-RT.15 by digesting the plasmid with XhoI and SalI andrecircularizing the plasmid. The resulting plasmid was designated aspRE1 -RT.

Expression of RT in E. coli CJ374 and BL21 harboring pRE1 -RT and pRE1-RT.15

The cultures were grown as described above. The soluble cell extract wasassayed for RT activity. Although the activity was extremely low, it wasclear that the RT activity in the induced cell extract was 10-timeshigher than that in the uninduced cell extract. The level of activitywas similar in both CJ374 and BL21. The levels of expression of RT frompRE1-RT and pRE1-RT.15 were similar.

Cloning of RT Gene under a Tac Promoter

Since RSV RT βp4 was not expressed well under a lambda pL promoter,expression was attempted under the control of a different promoter. TheRSV RT βp4 gene with and without the p15 gene was cloned under a tacpromoter. For cloning the RT gene, pRE1-RT was digested with NsiI,blunt-ended with T4 DNA polymerase and finally, digested with XhoI. TheRT fragment was purified from an agarose gel. For cloning the RT.15gene, pRE1-RT.15 was digested with NsiI, blunt-ended with T4 DNApolymerase and finally, digested with SalI. The RT.15 gene combinationfragment was purified from an agarose gel. The vector pTrc99A(Pharmacia) was digested with NcoI, blunt-ended with Klenow fragment andfinally, digested with SalI. The large vector fragment was purified andligated with either purified RT or RT.15 fragment. The resultingconstructs were introduced into E. coli DH10B. Clones with correctinserts were saved. The clones were designated as pTrcRT and pTrcRT.15.

Expression of RT under a Tac Promoter

E. coli cells harboring pTrcRT or pTrcRT.15 were grown at 37° C. in abuffered-rich medium to an A590 to 0.1 or 0.6 before addition of IPTG (1mM) for induction of RSV RT βp4 expression. The cells were collected 2hr after induction. The uninduced cultures were grown similarly withoutaddition of any inducer. The RT enzyme activity in soluble cell extractwas equivalent to that obtained from constructs with the lambda pLpromoter.

SDS Polyacrylamide Gel Electrophoresis of the Expressed Proteins

RSV RT is composed of two subunits of molecular mass 94 kD (β) and 62 kD(α). The α subunit is a proteolytic fragment derived from the β subunit.The proteolysis is accomplished by RSV p15 protease in vivo. However,when induced E. coli extracts bearing the plasmids described wereexamined, two induced proteins of molecular mass 75 kD and 62 kD weredetected. Both proteins were found to be mainly in the insolublefraction of E. coli extracts. The presence of a 75 kD and not theexpected 94 kD protein following expression in E. coli suggested that(i) there was a mutation near the carboxy end causing prematuretranslation termination, (ii) there was proteolysis in the E. colicytosol, or (iii) there was internal translation initiation of RT. Whenthe carboxy terminal region of the RT gene was sequenced, no mutationwas found that might have caused premature translation termination. Totest whether there was an internal translation start, the RT gene wascloned as a HpaI-XhoI fragment in pTrc99 digested with SmaI and SalI. Noinduced protein could be detected suggesting that there was no internalstart to generate either a 75 kD or 62 kD protein.

Expression of RT in a Variety of E. coli Hosts

As described above, expression of RSV RT was examined in E. coli DH10B,CJ374 and BL21. None of these E. coli hosts produced αβ RT, and thelevel of RT activity in each host was extremely low. Since M-MLV RTexpresses well in E. coli N4830 and HIV RT expresses well in E. coliRR1, the levels of expression of RSV RT in these two hosts wereexamined. Neither of these hosts was found to express active RT anybetter than the other hosts tested, nor did they produce any full length94 kD protein.

Expression of RT as a Fusion Protein

It is now well established that some proteins that do not express wellin E. coli are better expressed as a translation fusion, in which theprotein from a well expressed gene forms the amino end of the fusionprotein. The gene of one such fusion partner, thioredoxin, is present ina vector called pTrxfus (Genetics Institute; Cambridge, Mass.). Thelevel of expression of RSV RT βp4 fused with thioredoxin was thereforeexamined.

In the course of expressing RSV RT in a baculovirus system, the RSV RTgene fragment was cloned as a SmaI-XhoI fragment in pBacPAK9 toconstruct pBacPak-RT (see below). To make the thioredoxin fusion,pBacPak-RT was digested with XmaI (same recognition as SmaI) and PstIand the RT fragment was purified on an agarose gel. The vector pTrxfuswas digested with XmaI and PstI and the large vector fragment waspurified. The purified fragments were ligated and E. coli CJ374 andGI724 or GI698 (Genetic Institute, Mass.) were transformed with theligated material. Clones with the correct insert were saved. Whencultures were induced and the cellular extracts were assayed for enzymeactivity, no RT activity was detected. Both induced cultures, however,produced a large amount of insoluble fusion protein of the expected sizeas judged by SDS-PAGE of the extracts. Other fusions were also tested.The RSV RT β gene was fused to the GST (glutathione S transferase) gene,and the RSV RT α gene was fused to the lambda CRO protein gene.Expression of either fusion from the trc promoter in E. coli straindH10B resulted in a large amount of insoluble protein of the appropriatemolecular weight, but little RT activity was detected in extracts ofinduced cells. An expression vector with both fusions (GST-beta andCRO-alpha) was constructed in which the fusions were co-expressed byinduction of the trc promoter. Co-expression of both fusions in E. colistrain dH10B resulted in large amounts of insoluble protein of theappropriate molecular weights, but little RT activity.

Cloning and Expression Separately of Genes Encoding RSV RT Subunits α,β, and βp4 in a Baculovirus System

Cloning of RSV RT βp4 in a Baculovirus System

To clone the RSV RT βp4 gene in the baculovirus transfer vector,pBacPAK9 (Clontech), a fragment of the RSV RT gene was generated by PCR.To facilitate this PCR, the following oligonucleotide was designed witha BamHI site (bold) and the ATG initiation codon at the beginning of theRSV βp4 gene:

Oligonucleotide #16 (SEQ ID NO:22)

5′-TAT TAG GAT CCC ATG ACT GTT GCG CTA CAT CTG GC-3′

Oligonucleotides #12 and #16 (SEQ ID NOs: 18 and 22, respectively) wereused for PCR using pJD100 as template. The PCR product was digested withBamHI and XbaI, and then ligated to pBacPAK9 digested with BamHI andXbaI. One of the clones, pBP-RT(PCR) (FIG. 43), was used for furthercloning. To reconstitute the entire RT gene, the small HpaI-XhoIfragment of pBP-RT(PCR) was replaced with the 2500 bp HpaI-XhoI fragmentof pRE1-RT.15. The reconstituted plasmid was designated as pBP-RT(ATG)(FIG. 44).

Cloning of p15 in pBacPAK-RT(ATG)

Placing the RSV p15 protease gene in pBP-RT(ATG) was achieved by athree-step cloning. First, a p15 gene fragment was subcloned frompUC19-p15 (see above) into pSport1 (LTI) as a KpnI-SalI fragment togenerate pSport-p15. Second, the p15 fragment was subcloned frompSport-p15 as a NotI-SmaI fragment into a baculovirus transfer vector,pAcUW43, to generate pAcUW43-p15. This cloning was done to introduce thep15 gene under a p10 promoter. Finally, the p15 gene including the p10promoter was subcloned from pAcUW43-p15 as a XhoI-NotI fragment intopBP-RT (ATG) to generate pBP-RT15(ATG) (FIG. 45).

Expressing RSV RT βp4 in Insect Cells

Insect cells (SF9) were transfected with a mixture of pBP-RT15(ATG) andlinearized baculovirus vector DNA (BaculoGold, Pharmingen). In thissystem, recombination between the pBP-RT15(ATG) plasmid and thebaculoviral DNA results in recombinant viral genomes with the RSV RT βp4gene downstream of the polyhedrin promoter. Recombinant virus wasisolated from the supernatant by standard techniques and viral stockswere made, one of which was chosen for further study. Insect cells wereinfected with pure viral stock and infected cells were harvested atvarious times after infection. RT activity was easily detectable in theinfected cells.

Generation of an RNase H− RSV RT

The generation of a mutation in the RNase H region of the RSV RT βp4gene is described in detail above in Example 1. To introduce themutation into pBP-RT(ATG), the HpaI-KpnI fragment of pBP-RT(ATG) wasreplaced with the HpaI-KpnI fragment of M13RTH− (FIG. 13). The newlyconstructed plasmid was designated as pBP-RT(H−) ATG. Insect Cells (Sf9)were transfected with a mixture of pBP-RT(H−) and linearized baculovirusvector DNA (BaculoGold, Pharmingen). Recombination between thepBP-RT(H−) plasmid and the baculoviral DNA results in recombinant viralgenomes with the RSV RT βH−P4 gene downstream of the polyhedrinpromotor. Recombinant virus was isolated from the supernatant bystandard techniques. Insect cells were infected with viral stock andinfected cells were harvested at various times after infection. RTactivity was easily detectable in the infected cells.

Attaching a Histidine Tag to RSV RT βp 4

pBP-RT(H−) was cleaved with BamHI and XbaI and the 0.9 kb fragment withthe amino end of the RSV RT βp4 gene was cloned into pFastBacHT at theBamHI-XbaI sites, creating (by translational fusion) a βp4 partialconstruct with a histidine tag at the amino end (pFBHTβP4). The 2 kbXbaI-XhoI fragment with the carboxy end of RSV RT βH−p4 from pBP-RT(H−)was inserted into the XbaI-XhoI sites of pFBHTβP4, creating pFBH-TβP4.pFBH-TβP4 was transformed into E. coli DH10Bac cells and the His-taggedRSV RT βH-p4 gene was transposed to bacmid. Bacmid DNA was isolated andtransfected into SF9 insect cells. Viral preparations from infected cellcultures were used to infect SF21 cells, and His-tagged RSV RT βH-p4from infected cells was isolated and characterized.

Transfection of Insect Cells

The transfection of Sf9 cells was done using Baculogold virus(Pharmingen, Calif.) and pBP-RT(H−)ATG to generate recombinant virus.Ten wells on two 6-well (60 mm) tissue culture plates were seeded 1×10⁶Sf9 cells (LTI). The cells were allowed to attach for 30 min at 27° C.While the cells were attaching, ten tubes each containing 200 μl ofSf-900II SFM medium (LTI) were set up. In tube 1, 500 ng of Baculogoldand 2 μg of pBP-RT(H−)ATG were added. In tube 2, 250 ng of Baculogoldand 1 μg of pBP-RT(H−)ATG; and tube 3, 125 ng of Baculogold and 500 ngof pBP-RT(H−)ATG were added. Tubes 6 through 9 contained 36 μl oflipofectin (LTI). Contents of tube 1, tube 2, tube 3, tube 4 and tube 5were transferred to tubes 6, 7, 8, 9 and 10, respectively. Into each ofthese tubes, 2 ml of Sf-900 II SFM were added. The culture medium fromeach well was removed and 2 ml of the DNA/lipofectin mix was dispensedin two wells, 1 ml each. Thus, the wells containing the mixture of tubes1 and 6, 2 and 7, and 3 and 8 contained DNA mixtures at differentamounts; the wells containing the mixtures of tubes 4 and 9 werecontrols containing lipofectin but no DNA; and the wells containing themixtures of tubes 5 and 10 were controls containing neither DNA norlipofectin. The plates were incubated at 27° C. for 4 hr, the medium wasremoved and replaced with 4 ml fresh Sf-900 II SFM, and the plates wereincubated for an additional 72 hrs at 27° C. The phage supernatants fromthe DNA containing wells were removed and marked as primary phagestocks. A second infection was done by infecting 1×10⁶ Sf9 cells with 1ml of the primary phage stock to amplify the recombinant phage. Theplates were incubated for 48 hrs at 27° C. and the phage stocks werecollected.

Injection of Insect Larvae

Trichoplusa ni larvae were injected with recombinant virus bearingpBP-RT(H−)ATG and the larvae were harvested as described (Medin, J. A.,et al., Methods in Molecular Biology 39:26 (1995)).

Expression of RSV RT αH− in a Baculovirus system

pDA (FIG. 19) was transformed into E. coli DH10Bac cells, the RT αH−gene was transposed to bacmid, and the bacmid DNA was purified andtransfected into SF21 insect cells. Viral preparations from infectedcell cultures were used to infect SF21 cells, and RSV RT αH− frominfected cells was isolated and characterized.

Expression of RSV RT βH-His in a Baculovirus System

pDABH-His (FIG. 21) was cleaved with RsrII and PstI, and the 2.6 kbfragment with the βH-His gene was cloned into the RsrII-PstI sites ofpFastBac. pFBBH-His was transformed into E. coli DH10Bac cells, the RTβH-His gene was transposed to bacmid, and the bacmid DNA was purifiedand transfected into SF21 insect cells. Viral preparations from infectedcell cultures were used to infect SF21 cells, and RSV RT βH-His frominfected cells was isolated and characterized.

Cloning and Expression of Genes Encoding RSV αβ RT in which thePolymerase Active Site is Mutated

Generation of RSV RTs Mutated in the Polymerase Domain

Alignment of the RSV RT peptide sequence with sequences from HIV RT,M-MLV RT, and sequences of other RT genes revealed the probable locationof one of the catalytic residues in the RSV RT polymerase domain, D110(aspartic acid reside at position number 110). According to theliterature, mutation of the corresponding amino acid in the larger chainof HIV RT from D (aspartate) to E (glutamic acid) resulted in nearlytotal loss of polymerase activity. Single strand DNA was isolated frompJB-His by infection of E. coli DH5αF′IQ/pJB-His cells with M13KO7, andthis DNA was mutated by site-directed mutagenesis (see Example 1 fordetailed protocol) with the following oligonucleotide:

Oligonucleotide #17 (SEQ ID NO:23)

5′- GCAATCCTTGAGCTCTAAGACCATCAGGG 3′

This oligonucleotide induces the mutation of the aspartate residue atposition #110 or the RSV RT catalytic domain to glutamate (D110E) andadds an SstI site (bold), forming plasmid pJBD110E-His (FIG. 48). Thismutated site was introduced into the RSV RT α gene by inserting the 460bp NheI-Eco47III fragment from pJBD110E-His into the 6.5 kb fragment ofNheI-Eco47III cleaved pDA, forming pDAD110E (FIG. 49). The D110Emutation was also introduced into the β gene by inserting the 460 bpNheI-Eco47III fragment into NheI-Eco47III cleaved pFBBH-His (FIG. 46),forming pFBBD110E-His (FIG. 50). The 2.6 kb RSV RT βD110E gene wascloned from pFBBD110E-His into pDABHis (FIG. 51) as a 2.6 kb RsrII-EcoRIfragment, replacing the RSV RT β-His gene, forming pDABD110EHis (FIG.52). pDABHis was cleaved with XhoI+PvuI, and the 4.6 kb fragment withthe β gene was joined to the 4.9 kb pDAD110E XhoI-PvuI fragment, formingpDAD110EBHis (FIG. 53). The 4.6 kb pDABD110EHisXhoI- PvuI fragment withthe βD110E gene was joined to the 4.9 kb pDAD110E XhoI-PvuI fragment,forming pDAD110 EBD110E (FIG. 54) (which has a his tag despite itstruncated name). pDAD110EBHis, pDABD110EHis, and pDAD110EBD110E weretransformed into E. coli DH10B-Bac, the RT genes were transposed to thebacmid, and the bacmid DNA was purified and transfected into SF21 insectcells. Viral preparations from infected cell cultures were used toinfect SF21 cells, and RSV RT from infected cells was isolated andcharacterized.

The three mutant plasmids are summarized in the following table, where“w.t. ” refers to the wild type amino acid at the 110 position (D,aspartic acid) and D110E refers to the mutation at the 110 position (Dto E, glutamic acid):

Plasmid α β pDABHis w.t w.t. pDABD110EHis w.t D110E pDAD110EBHis D110Ew.t. pDAD110EB110E D110E D110E

Cloning and Expression of RSV βp4 RT in Yeast

Cloning of the RSV βp4 RT Gene

The pHIL-D2 vector available from InVitrogen (CA) was digested withEcoRI, blunt ended with Klenow fragment and treated with alkalinephosphatase. pRE1-RT.15 was digested with NdeI and blunt ended withKlenow fragment. The NdeI digest generated the entire RSV RT genewithout the p15 protease gene. The fragments were ligated and E. coliDH10B was transformed with ligation mixture. The correct clones wereselected for proper insert and orientation. Two of the 8 clones testedhad the RT gene fragment in the correct orientation. One of the clones,pHILD2-RT, was used for further experimentation. To introduce the RNaseH− domain in this plasmid, pHILD2-RT was digested with BamHI plus XhoIand the wild type fragment was replaced with BamHI-XhoI fragment frompBacPAK-RT(H−)ATG. The final clone was screened with SstII. The clonewas designated as pHILD2-RT(H−).

Transformation of Pichia pastoris

Pichia pastoris GS115 (InVitrogen) was used for transformation accordingto the protocol recommended by InVitrogen. The plasmids, pHILD2-RT andpHILD2RT(H−), were digested with NotI, phenol-chloroform extracted andethanol precipitated before transformation. The transformation yielded20 clones for wild type RSV RT and 12 clones for RSV H− RT in theregeneration plates. These clones were screened for their growth inmethanol containing plates. Two putative clones were selected frominitial 20 clones (wild type RT). One of these two, H1, was completelyincapable of growing in methanol, and the other, H2, was capable ofgrowing very slowly in methanol. For the RSV H− RT clones, three cloneswere selected out of 12 screened. One of the clones, H-3, grew veryslowly in methanol. The others, H-4 and H-5, grew moderately inmethanol. Clones H1 and H3 were chosen for expression studies.

RSV βp4 RT Expression in Pichia pastoris

Clones H1 and H3 were grown and induced essentially as described in theuser's manual provided by the manufacturer (InVitrogen). As a control,GS115 containing a β-galactosidase gene (InVitrogen) was grown andinduced side by side. While no RT activity could be detected in theGS115/β-gal cells, an appreciable level of activity was detected in bothH1 (wild type RT) and H3 (H− RT) cells. In addition, the activityincreased with increased time of induction.

Isolation of RSV ββ, α and βp4βp4

RSV ββ RT Isolation

RSV ββ RT was purified as described in Example 2 for RSV αβ RT with thefollowing exceptions. RSV ββ RT eluted from the AF-Heparin-650M columnfrom 55-62% Buffer T, and from the Mono S HR 5/5 column from 58-62%Buffer J. RSV ββ RT was 90% homogeneous as judged by SDS-PAGE.

RSV α RT Isolation

RSV α RT was purified as described in Example 2 for RSV αβ RT with thefollowing exceptions. The Chelating Sepharose Fast Flow column wasequilibrated with 100% Buffer A and washed with 100% Buffer A after theclarified crude extract was passed over the column. RSV α RT was elutedwith a 10-column volume linear gradient of 100% Buffer A to 75% BufferA+25% Buffer B. The peak fractions of RT activity from the ChelatingSepharose Fast Flow column (10-12% Buffer B) were pooled, dithiothreitoland EDTA were added to the pool to achieve final concentrations of 1 mMand 0.1 mM, respectively, and the pool was dialyzed overnight against95% Buffer S+5% Buffer T. The dialyzed pool was loaded on a 22 mlAF-Heparin-650M column equilibrated in Buffer S. After a wash with 9column volumes of 95% Buffer S+5% Buffer T, the column was eluted with a9 column volume linear gradient of 95% Buffer S+5% Buffer T to 60%Buffer S+40% Buffer T. The peak fractions of RT activity (11-20% BufferJ) were pooled and dialyzed for 3 to 4 hours against 97.5% Buffer H and2.5% Buffer J. The dialyzed pool was loaded unto a 3.5 mlphosphocellulose column (Whatman) equilibrated in 100% Buffer H. After awash with 12 column volumes of 97.5% Buffer H+2.5 Buffer J, the columnwas eluted with a 14 column volume linear gradient of 97.5% BufferH+2.5% Buffer J to 60% Buffer H+40% Buffer J. The peak fractions of RTactivity (12-20% Buffer J) were pooled and dialyzed overnight against99% Buffer S+1% Buffer T. The dialyzed pool was loaded unto a Mono S HR5/5 column equilibrated in Buffer S. After a wash with 10 column volumesof 100% Buffer S, the column was eluted with a 20 column volume lineargradient of 100% Buffer S to 75% Buffer S+25% Buffer T. The RT peakfractions (15-17% Buffer T) were pooled, dialyzed against Storage Bufferovernight, and stored at −20° C. RSV α RT was found to be 80%homogeneous as judged by SDS-PAGE.

RSV βp4βp4 RT Isolation

RSV βp4βp4 RT was purified as described in Example 2 for RSV αβ with thefollowing exceptions. The pooled RT fractions from the ChelatingSepharose Fast Flow column were dialyzed overnight against 90% BufferH+10% Buffer J. The RSV βp4βp4 RT precipitated in this buffer. The RTwas recovered by centrifugation, dissolved in Storage Buffer containing0.5 M KCl, and stored at −20° C. RSV βp4βp4 RT was found to be >95%homogeneous as judged by SDS-PAGE.

Estimation of the Amounts of α, αβ, and ββ RSV RT inBaculovirus-Infected Insect Cells

Chromatography methods described in Example 2 can be used to separateand isolate any α, αβ and ββ RSV RT present in crude extracts fromvirus-infected insect cells. RSV RT α is separable from the other twoenzymes forms by chromatography on a Chelating Sepharose Fast Flowcolumn, where α either does not bind or elutes at <30 mM imidazole, andαβ and ββ elute together at >50 mM imidazole. This is possible by virtueof the His6 tag present on β, but not on α. RSV αβ and ββ RT areseparable subsequently by chromatography on Heparin-650M (αβ elutes at0.45 M KCl and ββ elutes at 0.58 M KCl). Reverse transcriptase isquantitated by assay with poly(C).oligo(dG) (Gerard, G. F., et al.,Biochemistry 13: 1632 (1974)). The specific activities of RSV RT α, αβand ββ with poly(C).oligo(dG) are 140,000, 90,000 and 6,000 units/mg ofprotein, respectively.

Cloning, Expression, Purification and Use of RSV Viral Protease p15

RSV viral protease was cloned and expressed as a linked dimer in E. coliand purified from inclusion bodies as described (Bizub, D., et al., J.Biol. Chem. 266:4951 (1991)).

Reaction mixtures (25 μl) used to digest RSV βp4 RT with RSV proteasecontained 100 mM NaPO₄ (pH 6.0 to 7.0), 1 mM 2-mercaptoethanol, 0.01%(W/V) Triton X-100, 2.4 M NaCl, 5 μg RSV βp4 RT, and 5 μg RSV protease.Incubations were for 1 to 16 hours at 4° C. Digestion products wereanalyzed by SDS-PAGE and by assay for recovery of RT activity.

Assay of RNase H Activity of RSV RT

The RNase H activity of RSV RT was determined by monitoring thesolubilization of[³H]poly(A) in [³H]poly(A).poly(dT). Reaction mixtures(50 μl) contained 50 mM Tris-HCl (pH 8.4), 20 mM KCl, 10 mM MgC1I, 10 μMeach of [³H] poly(A) (300 cpm/pmole) and poly(dT) in[³H]poly(A).poly(dT) and 10 mM dithiothreitol. Reaction mixtures wereincubated at 37° C. for 20 minutes. Incubations were terminated by theaddition of 80 μl of 20% (W/V) TCA and 10 μl of 1 mg/ml tRNA, and aftercentrifugation the amount of [³H]poly(A) solubilized was determined bycounting the supernatant in aqueous scintillation fluid. One unit ofRNase H activity was the amount of enzyme that solubilized one nmole of[³H]poly(A) in 20 minutes at 37° C.

Assay of DNA Polymerase Activity with Poly(C).Oligo(dG)₁₂₋₁₈

The RNA-dependent DNA polymerase activity of RSV RT was determined bymonitoring the synthesis of acid insoluble [³H]poly(dG) formpoly(C).oligo(dG). Reaction mixtures (50 μl) contained 50 mM Tris-HCl(pH 8.4), 50 mM KCl, 10 mM MgCl₂, 0.5 mM poly(C), 0.2 mM oligo(dG)₁₂₋₁₈,0.5 mM [³H]dGTP (40 cpm/pmole), and 10 mM dithiothreitol. Reactions wereincubated at 37° C. for 10 minutes and labeled products were acidprecipitated on GF/C glass filters that were counted in a scintillationcounter. One unit of DNA polymerase activity was the amount of enzymethat incorporated one nmole of [³H]dGTP in 10 minutes at 37° C.

Results and Discussion

Alternative Methods of Generating RSV αβ RT

Examples 1 through 6 demonstrated that RNase H⁻ forms of avian RT aremore efficient than RNase H⁺ RT in copying mRNA. The studies presentedin this Example were designed to determine the efficiency of mRNAcopying by RSV RNase H⁻ αβ RT that was generated by co-expression of theRSV α and β genes in several expression systems.

Expression in E.coli

SmaIl amounts of soluble and active α, ββ, βp4βp4, and αβ RSV RT havebeen purified from E.coli (Alexander, F., et al., J. Virol. 61: 534(1987); Weis, J. H., and Salstrom, J. S., U.S. Pat. No. 4,663,290(1987); Soltis, D. A. and Skalka, A. M., Proc. Natl. Acad. Sci. USA85:3372 (1988); and Cherhov, A. P., et al. Biomed Sci. 2: 49 (1991)).However, most of the RSV RT expressed in E. coli in these previousreports was in an insoluble form.

The present efforts to express amounts of RSV RT proteins easilypurified from E.coli are documented in the Materials and Methods sectionof this Example. In general, similar results to those previouslypublished were obtained; that is, most of the RSV RT protein expressedin E. coli was insoluble, and only small amounts of RT activity wereobserved. Because of low RT levels, no attempts were made to purify RSVRT expressed in E. coli.

Expression of the RSV RT βp4 or β Gene in Cultural Insect Cells

Heterodimeric p66/p51 HIV RT has been produced in E. coli and yeast hostcells when the cloned gene for HIV p66 was expressed (Lowe, D. M., etal., Biochemistry 27:8884 (1988); Muller, B., et al., J. Biol. Chem.264:13975 (1989); and Barr, P. J., et al., BioTechnology 5:486 (1987)).Formation of the heterodimer occurs by proteolytic processing of p66/p66by endogenous host proteases. In contrast, expression of the gene forHIV RT p66 in cultured insect cells yielded only p66/p66 homodimer(Kawa, S., et al., Prot. Expression and Purification 4:298 (1993)).

In the present studies, expression of the gene for RSV RT βp4 incultured insect cells was similarly found to result exclusively in theproduction of homodimer βp4βp4 (data not shown); little processing to αβor α was observed. When the RSV RT β gene was expressed in these cells,however, all three forms of RSV Rt were produced (Table 6). Most of theRT present in the cells was ββ (50-80%); a small amount of αβ wasproduced (˜10%); and even α was obtained (10-40%). These results suggestthat endogenous host proteases in cultured insect cells proteolyze ββ,but not βp4βp4, to αβ. The RSV αβ RT generated by proteolysis of ββ hadmuch lower functional activity than αβ generated by co-expression of theRSV RT α and β genes (Table 7).

TABLE 6 Expression Levels of RSV RTs in Insect Cells Infected With RSVRT β Gene Amount of RT Present per 30 grams of Insect Cells RSV ββ RTRSV αβ RT RSV α RT Infection No. mg % mg % mg % 1 0.7 54 0.1 8 0.5 38 20.12 79 0.017 11 0.015 10 3 0.55 83 0.05 8 0.06 9

Expression of RSV RT βp4 in Insect Larvae

Baculovirus bearing the RSV RT βp4 was also used to infect live larvaeby physical injection of virus. The level of proteases present in larvaeand in larval extracts is much higher than in cultured insect cells.Processing of βp4 to multiple forms of proteolyzed RT was observed,including processing to α. The major species of RT that could bepurified from these extracts had four major bands that migrated onSDS-PAGE at 97 kDa (His-tagged β), 87 kDa (proteolyzed β), 67 kDa(partially processed His-tagged α), and 62 kDa (α with His tag removedby proteolysis). This RSV RT had a specific activity of 55,000 units/mgprotein, comparable to RSV αβ RT. In the functional activity assay(Example 3), however, the RT purified from larvae had 85% and 60% of thetotal product and full length product functional activity, respectively,of RSV αβ RT generated by co-expression of α and β (Table 7).

TABLE 7 Specific and Functional Activities of Various Forms of RSV RTsExpressed in Insect Cells RT Form Isolated RSV αβ RT RSV ββ RT RSV α RTRSV βp4βp4 RT Specific Functional Activity^(b) Specific FunctionalActivity Specific Functional Activity Spec. Functional Activity Gene(s)Activity^(a) Total Full-length Activity Total Full-length Activity TotalFull-length Act. Total Full-length Expressed (U/mg) (ng/μg)^(c)(ng/μg)^(d) (U/mg) (ng/μg) (ng/μg) (U/mg) (ng/μg) (ng/μg) (U/mg) (ng/μg)(ng/μg) α and β 53,191 4,092 874 NP^(e) NP NP ND^(f) ND ND NP NP NP β25,113 1,098 116 15,819 584 41 ND ND ND NP NP NP βp4 NP NP NP NP NP NPNP NP NP 15,984 450 67 α NP NP NP NP NP NP 48,759 272 6 NP NP NP^(a)Specific activity = units (U) RT activity/mg RT protein inpoly(A)·oligo(dT) assay (Houts et al., J. Virol. 29:517 (1979)).^(b)Functional activity established with 7.5 Kb RNA as described inExample 3. ^(c)Mass of total reverse transcribed product, ng of productproduced per μg of RT used. ^(d)Mass of full-length reverse transcribedproduct, ng of product produced per μg of RT used. ^(e)“NP” = RT formnot produced by insect cells expressing the indicated gene. ^(f)“ND” =RT form produced by insect cells, but not analyzed in present studies.

Co-expression of RSV RT βp4 and RSV Protease p15 in Cultured InsectCells

Heterodimeric p66/p51 HIV RT has been produced efficiently in E. coli byco-expression of HIV protease and HIV RT p66 (Mizuahi, V., et al., Arch.Biochem Biophys. 273:347 (1989) and Le Grice, S. F. J. andGruninger-Leitch, F., Eur. J. Biochem. 187:307 (1990)). Co-expression ofthe viral protease increased the overall efficiency of convertingp66/p66 to heterodimner. As described above in the Materials and Methodssection of this Example, co-expression of RSV RT β and RSV protease p15genes in E. coli did not result in enhanced production of RSV αβ RT.Similarly, co-expression of RSV RT βp4 and RSV protease p15 in culturedinsect cells did not appreciably enhance the formation of RSV αβ RT.

Processing of RSV RT βp4 with RSV Protease p15 in vitro

Heterodimeric p66/p51 HIV RT has been produced from p66 purified from E.coli by treatment with HIV protease in vitro (Chattopodhyay, D., et al.,J. Biol. Chem. 267:14227 (1992)). Purified RSV RT βp4 was treated withRSV protease p15 to generate RSV αβ RT. This approach was successful ingenerating some αβ from βp4 based upon SDS-PAGE analysis, but severaldifficulties were encountered. First, contrary to what was observed withviral protease treatment of HIV p66 RT, processing of βp4 to αβ did notalways stop at αβ, as α in excess of β was formed as proteolysisproceeded. Second, significant loss of DNA polymerase activity wasobserved during proteolysis, suggesting RSV RT was partially inactivatedby the acid pH reaction conditions required by RSV protease.

Processing of RSV RT βp4 with Chymotrypsin in vitro

Heterodimeric p66/p51 HIV RT has also been produced from p66 by limitedproteolysis with α-chymotrypsin (Lowe, D. M., et al., Biochemistry27:8884 (1988)). This approach was tried unsuccessfully with RSV RT βp4.We found the digestion of βp4 with α-chymotrypsin was difficult tocontrol, and proteolysis was observed to not stop at αβ, but to proceedto conversion of βp4 to α.

Mixing of RSV RT α and β (in vitro) to Generate αβ

Heterodimeric p66/p51 HIV RT has been produced by mixing separate crudecell lysates containing p51 alone and p66 alone (Stahlhut, M., et al.,Protein Expression and Purification 5:614 (1994)). Mixing of theseparate subunits results in formation of a 1:1 molar complex ofp66/p51. In contrast, mixing of purified RSV α RT with purified RSV β RTat approximately a 1:1 molar ratio did not result in the formation of anαβ complex. These results are consistent with the notion that the RSVsubunits, once folded separately in an active conformation, prefer toremain separate when mixed.

Relative Ability of Various Forms of RSV RT to Copy RNA

A comparison was made of the ability of four different forms of RSV RT(αβ, ββ, α, and βp4βp4) to copy RNA. The RNase H active site of eachsubunit in these enzymes was mutated to eliminate RNase H activity. EachRT was expressed in cultured insect cells and purified by methodsdescribed above and in Example 1. Two RNAs were used for comparison:synthetic homopolymer poly(A) and 7.5-Kb mRNA. With poly(A).oligo(dT) astemplate-primer, a specific activity was calculated by determining aninitial rate of poly(dT) synthesis catalyzed at limiting enzymeconcentration, and then normalizing the rate to a given mass (mg) of RTin the reaction. This specific activity simply represents the ability ofa given RT to incorporate a single deoxynucleotide with an artificialtemplate, and does not necessarily represent the ability of the enzymeto copy heteropolymeric RNA. With 7.5-Kb RNA as template, the ability ofthe RTs to make a full-length copy of a long heteropolymeric RNA wasassessed (see Example 3 for details). The results are shown above inTable 7.

Two different forms of αβ were characterized in Table 7. One form wasgenerated as the result of the expression of the RSV RT β gene andsubsequent proteolytic processing in host insect cells, and had reducedspecific and functional activity. The other form of αβ was generated byco-expression of the RSV RT α and β genes. This form of αβ had a similarspecific activity to α, approximately 50,000 units/mg, and had a higherspecific activity than either ββ or βp4βp4 (approximately 16,000units/mg). Comparison of the functional activity of this αβ to other RTforms showed a much more dramatic contrast. RSV αβ RT produced 7, 9 and15 times more total cDNA per mass of enzyme than ββ, βp4βp4 and α,respectively, from 7.5-Kb RNA. Even greater differences were observedwhen yield of full length product was assessed: RSV αβ RT produced 21,13 and 146 times more full length product per mass of enzyme than ββ,βp4βp4 and α, respectively. RSV αβ RT produced by co-expression of theRSV RT α H⁻ and βH⁻ genes is therefore much more efficient in copyingmRNA than is any other form of RSV RT prepared by analogous methods.

Evidence that only the α Subunit in RSV αβ RT Is Active

Selective DNA polymerase active site mutagenesis of the HIV RTheterodimer p66/p51 has shown that only the DNA polymerase active siteof p66 is crucial for DNA polymerase activity (LeGrice, S. F. J., etal., The EMBO Journal 10: 3905 (1991) and Hostomsly, Z., et al., J.Virol. 66: 3179 (1992)). The p51 subunit in the HIV p66/p51 heterodimerapparently assumes a conformation which does not have a substratebinding cleft and therefore does not participate directly in dNTPbinding and incorporation (Jacob-Molina, A., et al., Proc Natl. Acad SciUSA 90: 6320 (1993)).

In the present studies, the same question was asked for RSV αβ RT. Inthis case, since each subunit contains both a DNA polymerase and RNase Hactive site, combinations of DNA polymerase mutants alone and RNase Hmutants alone were characterized. The results are shown in Table 8.

TABLE 8 DNA Polymerase and RNase H Activities of Various Forms of RSVRT. Specific Activity (Units/mg) Ratio of DNA RSV RT Form DNA PolymeraseRNase H Polymerase/RNase H αH⁺/βH⁺ 52,095 924 56.4 αH⁻/βH⁻ 53,191 <0.1 —αH⁺/βH⁻ 48,250 760 64.5 αH⁻/βH⁺ 50,909 4.5 11,313 βH⁻/βH⁻ 15,819 <0.1 —αH⁻ 48,759 <0.1 — αpol⁺/βpol⁺ 52,095 924 56.4 αpol⁻/βpol⁻ 50 600 0.08αpol⁺/βpol⁻ 57,500 900 63.9 αpol⁻/βpol⁺ 1,143 629 1.82

As shown in Table 8, when the RNase H active site in both subunits wasmutated to eliminate RNase H activity, RNaseH activity in purifiedenzyme was reduced to below detectable levels, while DNA polymeraseactivity was unchanged. When the RNase H active site in β was alteredwith the same mutation and the α RNase H active site was wild type, theenzyme was similar to wild type in polymerase and RNase H activity. Incontrast, mutation of the α subunit in RNase H, but not the β subunit,resulted in a 200-fold reduction in RNase H activity. Therefore, in thecase of RSV αβ RT, the RNase H domain of the large subunit (β) folds inan inactive conformation. Examination of DNA polymerase active sitemutants in RSV αβ RT revealed the same results (Table 8). The α subunit,but not the β subunit, supplies the DNA polymerase catalytic activity.So, in contrast to HIV p66/p51 RT, the smaller RSV αβ RT subunit, notthe larger, maintains enzymatic activity. The common element in bothenzymes is that the subunit possessing DNA polymerase and RNase Hdomains folds in an active conformation, while subunits missing theRNase H domain or possessing an additional domain (integrase), fold inan inactive conformation.

In sum, these results demonstrate that only the αβ form of ASLV RTcopies mRNA efficiently. In addition, the present results indicate thatexpression of ASLV RT in E. coli results in little or no production ofactive RT, while expression and activity increase dramatically bycloning and expression of ASLV RT in insect cells and yeast. Finally, byplacing point mutations in either the DNA polymerase or the RNase Hactive site of the α or β subunit of RSV ad RT, it has now beendiscovered that the DNA polymerase and RNase H catalytic activities ofthe RSV RT αβ heterodimer reside in the α subunit alone.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

24 1 39 DNA Artificial Sequence Oligonucleotide 1 auggagaucu cucatatgactgttgcgcta catctggct 39 2 37 DNA Artificial Sequence Oligonucleotide 2aacgcguacu agugttaaca gcgcgcaaat catgcag 37 3 36 DNA Artificial SequenceOligonucleotide 3 cuacuacuac uaggtaccct ctcgaaaagt taaacc 36 4 45 DNAArtificial Sequence Oligonucleotide 4 caucaucauc auctcgagtt atgcaaaaagagggctcgcc tcatc 45 5 36 DNA Artificial Sequence Oligonucleotide 5ggacccactg tctttaccgc ggcctcctca agcacc 36 6 39 DNA Artificial SequenceOligonucleotide 6 caucaucauc aucccgggtt aatacgcttg gaaggtggc 39 7 35 DNAArtificial Sequence Oligonucleotide 7 cuacuacuac uatcatgact gttgcgctacatctg 35 8 33 DNA Artificial Sequence Oligonucleotide 8 cuacuacuacuaggtaccct ctcgaaaagt taa 33 9 52 DNA Artificial SequenceOligonucleotide 9 caucaucauc augaggaatt cagtgatggt gatggtgatg tgcaaaaagagg 52 10 41 DNA Artificial Sequence Oligonucleotide 10 actggaattcatgccaatcc atcaccatca ccatcacccg t 41 11 41 DNA Artificial SequenceOligonucleotide 11 acgtgtcgac catatggatg actaggtgaa acgggtgatg g 41 1271 DNA Artificial Sequence Annealed primer product 12 actggaattcatgccaatcc atcaccatca ccatcacccg tttcacctag tcatccatat 60 ggtcgacacg t71 13 48 DNA Artificial Sequence Oligonucleotide 13 gactagttctagatcgcgag cggccgccca ttaactctcg ttggcagc 48 14 16 DNA ArtificialSequence Oligonucleotide 14 tcgacccacg cgtccg 16 15 12 DNA ArtificialSequence Oligonucleotide 15 cggacgcgtg gg 12 16 42 DNA ArtificialSequence Oligonucleotide 16 auggagaucu cugaattcat gactgttgcg ctacatctggct 42 17 32 DNA Artificial Sequence Oligonucleotide 17 attattcatatgactgttgc gctacatctg gc 32 18 25 DNA Artificial SequenceOligonucleotide 18 tacgatctct ctccaggcca ttttc 25 19 24 DNA ArtificialSequence Oligonucleotide 19 actcgagcag cccgggaacc tttg 24 20 48 DNAArtificial Sequence Oligonucleotide 20 attacccggg aggatatcat atgttagcgatgacaatgga acataaag 48 21 36 DNA Artificial Sequence Oligonucleotide 21atatgtcgac tcacagtggc cctccctata aatttg 36 22 35 DNA Artificial SequenceOligonucleotide 22 tattaggatc ccatgactgt tgcgctacat ctggc 35 23 29 DNAArtificial Sequence Oligonucleotide 23 gcaatccttg agctctaaga ccatcaggg29 24 36 DNA Artificial Sequence Oligonucleotide 24 ggacccactgtctttaccgc ggcctcctca agcacc 36

What is claimed is:
 1. A composition for use in reverse transcription ofa nucleic acid molecule, said composition comprising two or more viralreverse transcriptases, or mutants or fragments thereof, or combinationthereof.
 2. The composition of claim 1, wherein said reversetranscriptase are retroviral reverse transcriptases.
 3. The compositionof claim 1, wherein said reverse transcriptases are selected from thegroup consisting of Moloney Murine Leukemia Virus (M-MLV), Rous SarcomaVirus (RSV), Avian Myeloblastosis Virus (AMV), Rous Associated Virus(RAV), Myeloblastosis Associated Virus (MAV), and Human ImmunodeficiencyVirus (HIV) reverse transcriptases.
 4. The composition of claim 1,wherein said reverse transcriptases or mutants or fragments comprise anα subunit, a β subunit, a βp4 subunit, or a combination thereof.
 5. Thecomposition of claim 1, wherein at least one of said mutants orfragments has substantially reduced RNase H activity compared to thecorresponding wild-type reverse transcriptase.
 6. The composition ofclaim 1, wherein at least one of said mutants or fragments lacks RNase Hactivity.
 7. The composition of any of claims 5 and 6, wherein at leastone of said mutants or fragments is selected from the group consistingof mutants or fragments of M-MLV, RSV, AMV, RAV, MAV, and HIV reversetranscriptases.
 8. The composition of any one of claims 5 and 6, whereinat least one of said mutants or fragments comprises an α subunit, a βsubunit, a βp4 subunit, or a combination thereof.
 9. The composition ofclaim 1, wherein said reverse transcriptases or mutants or fragments arepresent in said composition at working concentrations.
 10. A kit for usein reverse transcription of a nucleic acid molecule, said kit comprisingtwo or more viral reverse transcriptases, or mutants thereof, orfragments thereof, or a combination thereof.
 11. The kit of claim 10wherein said reverse transcriptases are retroviral reversetranscriptases.
 12. The kit of claim 10, wherein said reversetranscriptases are selected from the group consisting of M-MLV, RSV,AMV, RAV, MAV, and HIV reverse transcriptases.
 13. The kit of claim 10,wherein said reverse transcriptases or mutants or fragments comprise anα subunit, a β subunit, a βp3 subunit, or a combination thereof.
 14. Thekit of claim 10, wherein at least one of said mutants or fragments hassubstantially reduced RNase H activity compared to the correspondingwild-type reverse transcriptase.
 15. The kit of claim 10, wherein atleast one of said mutants or fragments lacks RNase H activity.
 16. Thekit of any one of claims 14 and 15, wherein at least one of said mutantsor fragments is selected from the group consisting of mutants orfragments of M-MLV, RSV, AMV, RAV, MAV, and HIV reverse transcriptases.17. The kit of any one of claims 14 and 15, wherein at least one of saidmutants or fragments comprises an α subunit, a β subunit, a βp4 subunit,or a combination thereof.
 18. The kit of claim 10, wherein said reversetranscriptases or mutants or fragments are present in said kit atworking concentrations.
 19. The kit of claim 10, said kit furthercomprising one or more components selected from the group consisting ofone or more nucleotides, one or more DNA polymerases, a suitable buffer,one or more primers and one or more terminating agents.
 20. The kit ofclaim 19, wherein said terminating agent is a dideoxynucleotide.
 21. Thekit of claim 19, wherein two or more of the components of said kit arepresent as a mixture or are present as separate components.